Antimicrobial cationic peptides and formulations thereof

ABSTRACT

Compositions and methods for making and using therapeutic formulations of antimicrobial cationic peptides are provided. The antimicrobial cationic peptide formulations may be used, for example, in the treatment of microorganism-caused infections, which infections may be systemic, such as a septicemia, or may be localized, such as in acne or an implanted or indwelling medical device.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Continuation of allowed U.S. patent applicationSer. No. 10/225,087, filed Aug. 20, 2002, which claims the benefit ofU.S. Provisional Patent Application No. 60/314,232 filed Aug. 21, 2001,where these applications are incorporated herein by reference in theirentireties.

TECHNICAL FIELD

The present invention relates generally to the treatment of infectiousdisease, and more specifically, to compositions comprising antimicrobialcationic peptides formulated for therapeutic use.

BACKGROUND OF THE INVENTION

During this century, modern society has been successful in controllinginfectious disease by, for example, using vaccines, using drugs (such asantibiotics), and using strict public health measures. These advanceshave been paralleled by successfully identifying the causative agents ofinfectious disease, which agents include bacteria, fungi, protozoa, andviruses. Thus, aside from a healthy host immune response, antibiotictherapeutic regimens now represent the primary course of treatment formost infectious diseases in developed countries. In contrast, infectiousdiseases remain a serious concern for developing countries, due to thelack of adequate sanitation and consequent poor hygiene, and forimmunocompromised individuals. However, due to the widespread use ofantibiotics, drug-resistance to one or more antibiotics is becoming anincreasingly common problem all over the world for controlling a numberof previously treatable infectious diseases (e.g., Staphylococcalinfections). Accordingly, treatment of nosocomial infections (i.e.,those arising in hospitals) and infections related to indwelling medicaldevices is becoming more difficult because of the intractable nature ofinfections due to drug-resistant microorganisms, which is a serious andworld-wide clinical concern.

A variety of artificial devices to assist in the performance of variousphysiological functions have been developed to be inserted into thehuman body for short periods, such as catheters, or to be insertedpermanently, such as artificial heart valves; however, the interfacebetween the device and body creates new biological conditions thatincrease the propensity of infection. For example, catheter-associatedinfections may have multiple potential sources of contaminants,including contaminants in the infusate that is directly injected,contaminants of the catheter hub where the administration set attachesto the catheter, contaminants carried hematogenously from remote sourcesof local infection to colonize the catheter, or contaminants ofcutaneous origin that invade the percutaneous tract extralumenally atthe time the catheter is inserted or in the days following insertion.Available evidence indicates that the majority of catheter-relatedbacteremias originate from the cutaneous microflora of the insertionsite. Given the evidence for the importance of cutaneous microorganismsin the pathogenesis of intravascular device-related infections, measuresto reduce colonization of the insertion site are of great importance inthe health care industry.

Another clinical indication of importance is nosocomial infections and,in particular, nosocomial pneumonia and nosocomial sinusitis.Contaminated secretions may be aspirated daily in the tracheobronchialtree, which may lead to pneumonia. Additionally, the risk of nosocomialpneumonia is increased after a tracheostomy is performed and duringprolonged endotracheal intubation. Sinusitis has been found to beassociated with an increased risk of nosocomial pneumonia, presumablydue aspiration of contaminated sinovial fluids into the distal airways.Sinusitis typically arises in the hospital setting among mechanicallyventilated patients. Recommended treatments for sinusitis of intubatedpatients, include removal of the tubes or systemic antibiotics. However,once again, the increase in antibiotic-resistant organisms makes thelatter treatment, whether preventative or curative, less efficacious.

Yet another clinical indication, although not life-threatening, is themost common skin disease of adolescence and early adulthood, acnevulgaris, or acne as it is generally called. In addition topsychological effects, such as anxiety, depression and withdrawal fromsociety, studies have also shown that acne vulgaris can directly andsignificantly affects a patient's quality of life. Antibiotic agentshave been extensively used for the treatment of acne for severaldecades; however, there is a growing concern that with the use ofantibiotics to treat acne, drug-resistant microorganisms will inevitablyemerge.

To address the issue of ever increasing drug-resistant microorganisms,investigations have turned to new classes of antibiotics, such asantimicrobial peptides. Antimicrobial peptides are found inevolutionarily diverse species including, for example, prokaryotes,plants, insects, and mammals. Antimicrobial peptides may be anionic, butmost known antimicrobial peptides are cationic. Multiple families ofantimicrobial cationic peptides are known and these peptides encompass awide variety of structural motifs, yet all of these cationic peptideshave similar physicochemical properties. For example, most knownantimicrobial cationic peptides are cationic at neutral pH, aregenerally less than 10 kDa, and are amphipathically “sided” in solutionsuch that hydrophobic side chains are regionalized. Many antimicrobialcationic peptides are known, including defensins, cecropins, melittins,magainins, indolicidins, and protegrins. The advantages of cationicpeptides are their ability to kill target cells rapidly, their broadspectrum of activity, and their activity against some of the moreserious antibiotic-resistant and clinically relevant pathogens. Mostimportantly, antimicrobial peptide-resistant microorganisms arerelatively difficulty to select in vitro. However, some antimicrobialpeptides have been found to be toxic (e.g., bee venom, wasp venom, andscorpion toxin), some have been found to have reduced activity in vivo(due to factors such as high mono- and divalent cation concentrations,polyanions, serum, apolipoprotein A-1, serpins, and proteases, althoughmany peptides are not affected by these factors), and some have beenfound to be less potent than conventional antibiotics.

Hence, a need exists for identifying modified or derivativeantimicrobial peptides with improved activity (and in some cases withreduced toxicity), for formulating such peptides and derivatives thereoffor optimal therapeutic use, and for developing therapeuticallyeffective clinical regimens for these cationic peptides. Furthermore,there is a need for formulations that are useful in a variety ofclinical indications. The present invention meets such needs, andfurther provides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides antimicrobial cationic peptides, inparticular indolicidin peptides and analogs or derivatives thereof, andformulations of such peptides for use in a variety of therapeuticsettings, such as in treating or preventing, for example, infectiousdisease associated with foreign bodies, primary infection sites, orsecondary infections arising from a primary disease state.

In one aspect, the present invention provides a composition thatcomprises an antimicrobial cationic peptide, a viscosity-increasingagent, and a solvent. In certain embodiments the solvent is water,glycerin, propylene glycol, isopropanol, ethanol, or methanol. Incertain other embodiments, the solvent is glycerin at a concentrationranging from about 0.1% to about 20% or from about 9% to about 11%. Instill other embodiments, the solvent is propylene glycol at aconcentration ranging from about 0.1% to about 20% or from about 9% toabout 11%. In yet other embodiments, the solvent comprises at least oneof water, glycerin, propylene glycol, isopropanol, ethanol, andmethanol. In further embodiments, the solvent comprises at least one ofwater at a concentration up to 99%, glycerin at a concentration up to20%, propylene glycol at a concentration up to 20%, ethanol at aconcentration up to 99%, and methanol at a concentration up to 99%.

In certain embodiments, the viscosity-increasing agent is dextran,polyvinylpyrrolidone, hydroxyethyl cellulose, or hydroxypropylmethylcellulose. In another embodiment, the viscosity-increasing agentis hydroxyethyl cellulose at a concentration ranging from about 0.5% toabout 5% or from about 1% to about 3%. In another embodiment, theviscosity-increasing agent is hydroxypropyl methylcellulose at aconcentration ranging from about 1% to about 3%. In other embodiments,the viscosity-increasing agent is dextran at a concentration rangingfrom about 0.1% to about 5% or from about 0.5% to about 1%. In certainother embodiments, where the viscosity-increasing agent is hydroxyethylcellulose, the composition further comprises a secondviscosity-increasing agent of dextran, polyvinylpyrrolidone, orhydroxypropyl methylcellulose. In one embodiment, the secondviscosity-increasing agent is polyvinylpyrrolidone. In relatedembodiments, the polyvinylpyrrolidone is at a concentration ranging fromabout 0.1% to about 5% or from about 0.5% to about 1%. In anotherembodiment, the second viscosity-increasing agent is hydroxypropylmethylcellulose. In related embodiments, the hydroxypropylmethylcellulose is at a concentration ranging from about 1% to about 3%.In certain other embodiments, where the viscosity-increasing agent ishydroxypropyl methylcellulose, the composition further comprises asecond viscosity-increasing agent of dextran, or dextran atconcentration ranging from about 0.1% to about 5% or from about 0.5% toabout 1%. In still other embodiments, where the viscosity-increasingagent is hydroxypropyl methylcellulose, the composition furthercomprises a second viscosity-increasing agent of polyvinylpyrrolidone,or polyvinylpyrrolidone at a concentration ranging from about 0.1% toabout 5% or from about 0.5% to about 1%. In certain embodiments, thefirst viscosity-increasing agent comprises hydroxyethyl cellulose at aconcentration up to about 3% and second viscosity-increasing agentcomprises hydroxypropyl methylcellulose at a concentration up to about3%.

In another embodiment, the present invention provides a compositioncomprising an antimicrobial cationic peptide, a viscosity-increasingagent, and a solvent, which further comprises a buffering agent. Incertain embodiments, the buffering agent is at a concentration rangingfrom about 1 mM to about 200 mM. In other embodiments, the bufferingagent may comprise a monocarboxylate or a dicarboxylate. In furtherembodiments, the buffering agent is acetate, fumarate, lactate,malonate, succinate, or tartrate. In yet another embodiment, thecomposition further comprising a buffering agent has a pH ranging fromabout 3 to about 8.

In still other embodiments, any of the aforementioned compositionsfurther comprise a humectant. In one embodiment, the humectant issorbitol or glycerol. In further embodiments, any of the aforementionedcompositions further comprise a preservative. In one embodiment, thepreservative comprises benzoic acid, benzyl alcohol, phenoxyethanol,methylparaben, propylparaben, or a combination thereof. As used herein,any reference to an acid may include a free acid, a salt, and any esterthereof. In other embodiments, any of the aforementioned compositionsfurther comprise a humectant and a preservative.

In certain embodiments, the antimicrobial cationic peptide is anindolicidin or an analog or derivative thereof in any one of theaforementioned compositions. In other embodiments, the cationic peptideis at a concentration ranging from about 0.01% to about 10% or fromabout 0.5% to about 1.5% in any one of the aforementioned compositions.In yet other embodiments, any one of the aforementioned compositionshaving a cationic peptide that is a peptide of up to 35 amino acids,comprising one of the following sequences: 11B7CN, 11B32CN, 11B36CN,11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN, 11F63CN,11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN, 11J02ACN,11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN, Nt-acryloyl-11B7CN,Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN.

In another aspect there is provided a composition comprising anantimicrobial cationic peptide, a viscosity-increasing agent, a solvent,a humectant, and a buffering agent. In one embodiment, the humectant issorbitol or glycerol. In certain embodiments, the buffering agent is ata concentration ranging from about 1 mM to about 200 mM. In otherembodiments, the buffering agent comprises a monocarboxylate or adicarboxylate. In further embodiments, the buffering agent is acetate,fumarate, lactate, malonate, succinate, or tartrate. In yet anotherembodiment, the composition has a pH ranging from about 3 to about 8. Inanother embodiment, the composition further comprises a preservative. Inone embodiment, the preservative comprises benzoic acid, benzyl alcohol,phenoxyethanol, methylparaben, propylparaben, or a combination thereof.In certain embodiments, the solvent is water, glycerin, propyleneglycol, isopropanol, ethanol, or methanol. In certain other embodiments,the solvent is glycerin at a concentration ranging from about 0.1% toabout 20% or from about 9% to about 11%. In still other embodiments, thesolvent is propylene glycol at a concentration ranging from about 0.1%to about 20% or from about 9% to about 11%. In yet other embodiments,the solvent comprises at least one of water, glycerin, propylene glycol,isopropanol, ethanol, and methanol. In further embodiments, the solventcomprises at least one of water at a concentration up to 99%, glycerinat a concentration up to 20%, propylene glycol at a concentration up to20%, ethanol at a concentration up to 99%, and methanol at aconcentration up to 99%.

In certain embodiments, the viscosity-increasing agent is dextran,polyvinylpyrrolidone, hydroxyethyl cellulose, or hydroxypropylmethylcellulose. In another embodiment, the viscosity-increasing agentis hydroxyethyl cellulose at a concentration ranging from about 0.5% toabout 5% or from about 1% to about 3%. In another embodiment, theviscosity-increasing agent is hydroxypropyl methylcellulose at aconcentration ranging from about 1% to about 3%. In other embodiments,the viscosity-increasing agent is dextran at a concentration rangingfrom about 0.1% to about 5% or from about 0.5% to about 1%. In certainother embodiments, where the viscosity-increasing agent is hydroxyethylcellulose, the composition further comprises a secondviscosity-increasing agent of dextran, polyvinylpyrrolidone, orhydroxypropyl methylcellulose. In one embodiment, the secondviscosity-increasing agent is polyvinylpyrrolidone. In relatedembodiments, the polyvinylpyrrolidone is at a concentration ranging fromabout 0.1% to about 5% or from about 0.5% to about 1%. In anotherembodiment, the second viscosity-increasing agent is hydroxypropylmethylcellulose. In related embodiments, the hydroxypropylmethylcellulose is at a concentration ranging from about 1% to about 3%.In certain other embodiments, where the viscosity-increasing agent ishydroxypropyl methylcellulose, the composition further comprises asecond viscosity-increasing agent of dextran, or dextran atconcentration ranging from about 0.1% to about 5% or from about 0.5% toabout 1%. In still other embodiments, where the viscosity-increasingagent is hydroxypropyl methylcellulose, the composition furthercomprises a second viscosity-increasing agent of polyvinylpyrrolidone,or polyvinylpyrrolidone at a concentration ranging from about 0.1% toabout 5% or from about 0.5% to about 1%.

In certain embodiments, the antimicrobial cationic peptide is anindolicidin or an analog or derivative thereof in any one of theaforementioned compositions. In other embodiments, the cationic peptideis at a concentration ranging from about 0.01% to about 10% or fromabout 0.5% to about 1.5% in any one of the aforementioned compositions.In yet other embodiments, the cationic peptide is a peptide of up to 35amino acids, comprising one of the following sequences: 11B7CN, 11B32CN,11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN,11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN,11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN; 11J68CN,Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN in anyone of the aforementioned compositions.

In still another aspect, the present invention provides a compositioncomprising an antimicrobial cationic peptide, a buffering agent, and asolvent. In other embodiments, the composition further comprises ahumectant. In one embodiment, the humectant is sorbitol or glycerol. Inanother embodiment, the composition further comprises a preservative. Inone embodiment, the preservative comprises benzoic acid, benzyl alcohol,phenoxyethanol, methylparaben, propylparaben, or a combination thereof.In yet other embodiments, the composition further comprises aviscosity-increasing agent of dextran, polyvinylpyrrolidone,hydroxyethyl cellulose, or hydroxypropyl methylcellulose. In anotherembodiment, the viscosity-increasing agent is hydroxyethyl cellulose ata concentration ranging from about 1% to about 3%. In anotherembodiment, the viscosity-increasing agent is hydroxypropylmethylcellulose at a concentration ranging from about 1% to about 3%. Incertain other embodiments, wherein the viscosity-increasing agent ishydroxyethyl cellulose, the composition further comprises a secondviscosity-increasing agent of hydroxypropyl methylcellulose. In anotherembodiment, the viscosity-increasing agent is hydroxyethyl cellulose ata concentration up to about 3% and the second viscosity-increasing agentis hydroxypropyl methylcellulose at a concentration up to about 3%. In afurther embodiment, the composition further comprises an acne medicamentof retinoid, vitamin D3, or corticosteroid, and analogues or derivativesthereof.

In certain embodiments the solvent is water, glycerin, propylene glycol,isopropanol, ethanol, or methanol. In certain embodiments, the solventis glycerin at a concentration ranging from about 9% to about 11%. Inanother embodiment, the solvent is propylene glycol at a concentrationranging from about 9% to about 11%. In yet other embodiments, thesolvent comprises at least one of water, glycerin, propylene glycol,isopropanol, ethanol, and methanol. In further embodiments, the solventcomprises at least one of water at a concentration up to 99%, glycerinat a concentration up to 20%, propylene glycol at a concentration up to20%, ethanol at a concentration up to 99%, and methanol at aconcentration up to 99%.

In certain embodiments, the buffering agent comprises a monocarboxylateor a dicarboxylate. In other embodiments, the buffering agent isacetate, fumarate, lactate, malonate, succinate, or tartrate. In yetanother embodiment, the composition has a pH ranging from about 3 toabout 8. In further embodiments, the buffering agent is at aconcentration ranging from about 1 mM to about 200 mM or from about 4 mMto about 6 mM.

In certain embodiments, the antimicrobial cationic peptide is anindolicidin or an analog or derivative thereof in any one of theaforementioned compositions. In other embodiments, the cationic peptideis at a concentration ranging from about 0.01% to about 10% or fromabout 0.5% to about 1.5% in any one of the aforementioned compositions.In yet other embodiments, the cationic peptide is a peptide of up to 35amino acids, comprising one of the following sequences: 11B7CN, 11B32CN,11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN,11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN,11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN,Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN in anyone of the aforementioned compositions.

In yet another aspect, the present invention provides a compositioncomprising an antimicrobial cationic peptide at a concentration rangingfrom about 0.01% to about 10%; a viscosity-increasing agent of dextran,polyvinylpyrrolidone, hydroxyethyl cellulose, or hydroxypropylmethylcellulose; and a solvent of water, glycerin, propylene glycol,isopropanol, ethanol, or methanol; at a pH ranging from about 3 to about8. In certain embodiments, the composition comprises hydroxyethylcellulose at a concentration ranging from about 1% to about 2%. In otherembodiments, the composition comprises glycerin at a concentrationranging from about 9% to about 11%. In another embodiment, thecomposition further comprises a buffering agent. In one embodiment, thecomposition further comprising the buffering agent has a pH ranging fromabout 3.5 to about 7. In other embodiments, the buffering agentcomprises a monocarboxylate or a dicarboxylate. In yet otherembodiments, the buffering agent is acetate, fumarate, lactate,malonate, succinate, or tartrate. In certain other embodiments, thebuffering agent is at a concentration ranging from about 1 mM to about200 mM or from about 4 mM to about 6 mM. In another embodiment, thecomposition further comprises a preservative. In one embodiment, thepreservative comprises benzoic acid, benzyl alcohol, phenoxyethanol,methylparaben, propylparaben, or a combination thereof.

In certain embodiments, the antimicrobial cationic peptide is anindolicidin or an analog or derivative thereof in any one of theaforementioned compositions. In other embodiments, the cationic peptideis a peptide of up to 35 amino acids, comprising one of the followingsequences: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN,11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN,11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN,11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CNin any one of the aforementioned compositions.

In a further aspect, the present invention provides a compositioncomprising (a) an antimicrobial cationic peptide wherein the cationicpeptide is a peptide of up to 35 amino acids comprising one of thefollowing: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN,11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN,11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN,11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, orNt-glucosyl-11J38CN; (b) a viscosity-increasing agent wherein theviscosity-increasing agent is hydroxyethyl cellulose at a concentrationof about 1.2% to about 1.8%; (c) a buffer wherein the buffer is lactateat a concentration ranging from about 4 mM to about 6 mM; (d) a solventwherein the solvent comprises glycerin at a concentration ranging fromabout 9% to about 11% and water at a concentration ranging from about85% to about 90%; and (e) a pH ranging from about 3.5 to about 7. Incertain embodiments, the cationic peptide is at a concentration rangingfrom about 0.8% to about 1.2%. In yet another embodiment, provided aremethods to reduce microflora, or to treat or prevent an infection, at atarget site, the target site may be skin, and the skin may furthercomprise acne. In another embodiment, the composition may be applied toa target site to treat or prevent or ameliorate inflammation, such asinflammation associated with acne (or with an implanted or indwellingmedical device).

Another aspect of the present invention is a composition, comprising (a)an antimicrobial cationic peptide wherein the cationic peptide is apeptide of up to 35 amino acids comprising one of the following: 11B7CN,11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN,11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN,11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11B68CN,Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN; (b) abuffer wherein the buffer is lactate at a concentration ranging fromabout 4 mM to about 6 mM; (c) a solvent wherein the solvent comprisesethanol at a concentration ranging from about 45% to about 55% and waterat a concentration ranging from about 44% to about 54%; and (d) a pHranging from about 3.5 to about 7. In certain embodiments, the cationicpeptide is at a concentration ranging from about 0.8% to about 1.2%. Inother embodiments, the composition may further comprise an acnemedicament such as retinoid, vitamin D3, or corticosteroid, and analogsor derivatives thereof. In yet another embodiment, provided are methodsto reduce microflora, or to treat or prevent an infection, at a targetsite, the target site may be skin, and the skin may further compriseacne. In another embodiment, the composition may be applied to a targetsite to treat or prevent or ameliorate inflammation, such asinflammation associated with acne (or with an implanted or indwellingmedical device).

In still another aspect, the present invention provides a compositioncomprising (a) an antimicrobial cationic peptide wherein the cationicpeptide is a peptide of up to 35 amino acids comprising one of thefollowing: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN,11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN,11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN,11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, orNt-glucosyl-11J38CN; (b) a viscosity-increasing agent wherein theviscosity-increasing agent is hydroxyethyl cellulose at a concentrationof about 1.2% to about 1.8%; (c) a solvent wherein the solvent comprisesglycerin at a concentration ranging from about 9% to about 11% and waterat a concentration ranging from about 85% to about 90%; (d) apreservative wherein the preservative is benzoic acid at a concentrationranging from about 20 mM to about 30 mM; and (e) a pH ranging from about3.5 to about 4.7. In certain embodiments, the cationic peptide is at aconcentration ranging from about 0.8% to about 1.2%, or ranging fromabout 2.5% to about 3.5%. In yet another embodiment, provided aremethods to reduce microflora, or to treat or prevent an infection, at atarget site, the target site may be skin, and the skin may furthercomprise acne. In another embodiment, the composition may be applied toa target site to treat or prevent or ameliorate inflammation, such asinflammation associated with acne (or with an implanted or indwellingmedical device).

In another aspect there is provided a method for reducing microflora ata target site, comprising applying to the target site a compositioncomprising an antimicrobial cationic peptide, a viscosity-increasingagent, and a solvent. In certain embodiments, the microflora is aprokaryotic organism, a eukaryotic organism, or a virus. In someembodiments the target site is skin and in others the skin furthercomprises acne. In other embodiments, the target site is a mucosa, andin still other embodiments the mucosa further comprises a nasal passage.In one embodiment the nasal passage is an anterior naris. In certainother embodiments, the method further comprises inserting a medicaldevice at the target site before or after applying the composition. Inyet another embodiment, the method further comprises applying thecomposition to the device prior to inserting the device at the targetsite. In one embodiment, the device comprises a catheter and anotherembodiment is a central venous catheter. In certain other embodiments,the catheter is a vascular dialysis catheter, a pulmonary arterycatheter, a peritoneal dialysis catheter, or an umbilical catheter.

In a further aspect, the present invention provides a method fortreating or preventing infection at a target site, comprising applyingto the target site a composition comprising a cationic peptide, aviscosity-increasing agent, and a solvent. In certain embodiments, theinfection is caused by a prokaryotic organism, a eukaryotic organism, ora virus. In other embodiments, the infection at a target site isassociated with a medical device at the target site. In furtherembodiments, the method comprises applying the composition prior to orafter inserting a medical device at the target. In one embodiment, thedevice comprises a catheter and another embodiment is a central venouscatheter. In certain other embodiments, the catheter is a vasculardialysis catheter, a pulmonary artery catheter, a peritoneal dialysiscatheter, or an umbilical catheter. In some embodiments the target siteis skin and in others the skin further comprises acne. In otherembodiments, the target site is a mucosa, and in still other embodimentsthe mucosa further comprises a nasal passage. In one embodiment thenasal passage is an anterior naris.

In yet another aspect there is provided a method treating or preventinginflammation at a target site, comprising applying to the target site acomposition comprising a cationic peptide, a viscosity-increasing agent,and a solvent. In one embodiment, the target site further comprises aninfection. In a further embodiment, the inflammation at the target siteis associated with a medical device. In further embodiments, the methodcomprises applying the composition prior to or after inserting a medicaldevice at the target. In one embodiment, the device comprises a catheterand another embodiment is a central venous catheter. In certain otherembodiments, the catheter is a vascular dialysis catheter, a pulmonaryartery catheter, a peritoneal dialysis catheter, or an umbilicalcatheter. In some embodiments the target site is skin and in others theskin further comprises acne. In other embodiments, the target site is amucosa, and in still other embodiments the mucosa further comprises anasal passage. In one embodiment the nasal passage is an anterior naris.

In yet another aspect, the invention provides a method for amelioratinginflammation at a target site, comprising applying to the target site acomposition comprising a cationic peptide, a viscosity-increasing agent,and a solvent. In one embodiment, the target site further comprises aninfection. In a further embodiment, the inflammation at the target siteis associated with a medical device. In further embodiments, the methodcomprises applying the composition prior to or after inserting a medicaldevice at the target. In one embodiment, the device comprises a catheterand another embodiment is a central venous catheter. In certain otherembodiments, the catheter is a vascular dialysis catheter, a pulmonaryartery catheter, a peritoneal dialysis catheter, or an umbilicalcatheter. In some embodiments the target site is skin and in others theskin further comprises acne. In other embodiments, the target site is amucosa, and in still other embodiments the mucosa further comprises anasal passage. In one embodiment the nasal passage is an anterior naris.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions, and aretherefore incorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of a bacterial challenge test of anantimicrobial cationic peptide (1%, gel) formulation, illustrating theantimicrobial effectiveness of the formulation when seeded with a largeinoculum of various bacteria and fungi.

FIG. 2 shows the results of a bacterial challenge test of anantimicrobial cationic peptide (1%, petrolatum) formulation illustratingthe antimicrobial effectiveness of the formulation.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides compositions and methodsfor using antimicrobial cationic peptides to treat and/or preventinfectious diseases. The invention, therefore, relates generally to thesurprising discovery that antimicrobial cationic peptides may beformulated at a clinically relevant concentration to maximize their invivo and in vitro stability, release half-life, and antimicrobialactivity. According to the present invention, formulations ofantimicrobial cationic peptides (e.g., indolicidins and derivatives oranalogs thereof), as described herein, provide novel and usefulcompositions for use in a variety of therapeutic settings (e.g., in thetreatment and prevention of nosocomial infections, acne, and infectionsassociated with intravascular penetration, such as in the use ofhypodermic needles and catheters). Discussed in more detail below arecationic peptides suitable for use within the present invention, as wellas representative formulations and therapeutic uses. Any concentrationranges recited herein are to be understood to include concentrations ofany integer within the range and fractions thereof, such as one tenthand one hundredth of an integer, unless otherwise indicated.

A. Antimicrobial Cationic Peptides

The present invention is directed generally to antimicrobial cationicpeptides, which may be produced by a variety of methods (e.g., chemicalor recombinant) for use in the formulations as described herein.Suitable antimicrobial cationic peptides include, but are not limitedto, naturally occurring cationic peptides, which have been isolated, andderivatives or analogs thereof. An “isolated peptide, polypeptide, orprotein” is an amino acid sequence that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, nucleicacid (DNA or RNA), or other proteinaceous impurities associated with thepolypeptide in nature. Preferably, the isolated polypeptide issufficiently pure for therapeutic use at the desired dose.

An antimicrobial cationic peptide of the present invention may be arecombinant peptide or a synthetic peptide, and is preferably arecombinant peptide.

Peptides may be synthesized by standard chemical methods, includingsynthesis by automated procedure. In general, peptide analogues aresynthesized based on the standard solid-phase Fmoc protection strategywith HATU as the coupling agent. The peptide is cleaved from thesolid-phase resin with trifluoroacetic acid containing appropriatescavengers, which also deprotects side chain functional groups. Crudepeptide is further purified using preparative reversed-phasechromatography. Other purification methods, such as partitionchromatography, gel filtration, gel electrophoresis, or ion-exchangechromatography may be used. Other synthesis techniques, known in theart, such as the tBoc protection strategy, or use of different couplingreagents or the like can be employed to produce equivalent peptides.Peptides may be synthesized as a linear molecule or as branchedmolecules. Branched peptides typically contain a core peptide thatprovides a number of attachment points for additional peptides. Lysineis most commonly used for the core peptide because it has one carboxylfunctional group and two (alpha and epsilon) amine functional groups.Other diamino acids can also be used. Preferably, either two or threelevels of geometrically branched lysines are used; these cores form atetrameric and octameric core structure, respectively (Tam, Proc. Natl.Acad. Sci. USA 85:5409, 1988).

An antimicrobial cationic peptide is a peptide that typically exhibits apositive charge at a pH ranging from about 3 to about 10 (i.e., has anisoelectric point of at least about 9), and contains at least one basicamino acid (e.g., arginine, lysine, histidine). In addition, anantimicrobial cationic peptide generally comprises an amino acidsequence having a molecular mass of about 0.5 kDa (i.e., approximatelyfive amino acids in length) to about 10 kDa (i.e., approximately 100amino acids in length), or a molecular mass of any integer, or fractionthereof (including a tenth and one hundredth of an integer), rangingfrom about 0.5 kDa to about 10 kDa. Preferably, an antimicrobialcationic peptide has a molecular mass ranging from about 0.5 kDa toabout 5 kDa (i.e., approximately from about 5 amino acids to about 45amino acids in length), more preferably from about 1 kDa to about 4 kDa(i.e., approximately from about 10 amino acids to about 35 amino acidsin length), and most preferably from about 1 kDa to about 2 kDa (i.e.,approximately from about 10 amino acids to about 18 amino acids inlength). In another preferred embodiment, the antimicrobial cationicpeptide is part of a larger peptide or polypeptide sequence having, forexample, a total of up to 100 amino acids, more preferably up to 50amino acids, even more preferably up to 35 amino acids, and mostpreferably up to 15 amino acids. The present invention contemplates anantimicrobial cationic peptide having an amino acid sequence of 5 to 100amino acids, with the number of amino acids making up the peptidesequence comprising any integer in that range. An antimicrobial cationicpeptide may exhibit antibacterial activity, anti-endotoxin activity,antifungal activity, antiparasite activity, antiviral activity,anticancer activity, anti-inflammatory activity, wound healing activity,and synergistic activity with other peptides or antimicrobial compounds,or a combination thereof.

Exemplary antimicrobial peptides include, but are not limited to,cecropins, normally made by lepidoptera (Steiner et al., Nature 292:246,1981) and diptera (Merrifield et al., Ciba Found. Symp. 186:5, 1994), byporcine intestine (Lee et al., Proc. Nat'l Acad. Sci. USA 86:9159,1989), by blood cells of a marine protochordate (Zhao et al., FEBS Lett.412:144, 1997); synthetic analogs of cecropin A, melittin, andcecropin-melittin chimeric peptides (Wade et al., Int. J. Pept. ProteinRes. 40:429, 1992); cecropin B analogs (Jaynes et al., Plant Sci. 89:43,1993); chimeric cecropin A/B hybrids (Düring, Mol. Breed. 2:297, 1996);magainins (Zasloff, Proc. Nat'l Acad. Sci. USA 84:5449, 1987);cathelin-associated antimicrobial peptides from leukocytes of humans,cattle, pigs, mice, rabbits, and sheep (Zanetti et al., FEBS Lett.374:1, 1995); vertebrate defensins, such as human neutrophil defensins[HNP 1-4]; paneth cell defensins of mouse and human small intestine(Oulette and Selsted, FASEB J. 10:1280, 1996; Porter et al., Infect.Immun. 65:2396, 1997); vertebrate β-defensins, such as HBD-1 of humanepithelial cells (Zhao et al., FEBS Lett. 368:331, 1995); HBD-2 ofinflamed human skin (Harder et al., Nature 387:861, 1997); bovineβ-defensins (Russell et al., Infect. Immun. 64:1565, 1996); plantdefensins, such as Rs-AFP1 of radish seeds (Fehlbaum et al., J. Biol.Chem. 269.33159, 1994); α- and β-thionins (Stuart et al., Cereal Chem.19:288, 1942; Bohlmann and Apel, Annu. Rev. Physiol. Plant Mol. Biol.42:227, 1991); γ-thionins (Broekaert et al., Plant Physiol. 108:1353,1995); the anti-fungal drosomycin (Fehlbaum et al., J. Biol. Chem.269:33159, 1994); apidaecins, produced by honey bee, bumble bee, cicadakiller, hornet, yellow jacket, and wasp (Casteels et al., J. Biol. Chem.269:26107, 1994; Levashina et al., Eur. J. Biochem. 233:694, 1995);cathelicidins, such as indolicidin and derivatives or analogues thereoffrom bovine neutrophils (Falla et al., J. Biol. Chem. 277:19298, 1996);bacteriocins, such as nisin (Delves-Broughton et al., Antonie vanLeeuwenhoek J. Microbiol. 69:193, 1996); and the protegrins andtachyplesins, which have antifungal, antibacterial, and antiviralactivities (Tamamura et al., Biochim. Biophys. Acta 1163:209, 1993;Aumelas et al., Eur. J. Biochem. 237:575, 1996; Iwanga et al., CibaFound. Symp. 186:160, 1994).

In certain embodiments, preferred antimicrobial cationic peptides of thepresent invention are indolicidins or analogs or derivatives thereof(see Table 2 of Example 1). Natural indolicidins may be isolated from avariety of organisms, and, for example, the indolicidin isolated frombovine neutrophils is a 13 amino acid peptide, which is tryptophan-richand amidated at the C-terminus (see Selsted et al., J. Biol. Chem.267:4292, 1992). As noted above, a preferred indolicidin or analog orderivative thereof comprises 5 to 45 amino acids, more preferably 7 to35 amino acids, even more preferably 8 to 25 amino acids, and mostpreferably 10 to 14 amino acids (see, e.g., Table 2). The indolicidinsor analogs or derivatives thereof of the present invention may be usedat a concentration ranging from about 0.01% to about 10%, preferablyfrom about 0.5% to about 5%, and more preferably from either about 1% toabout 3% or about 4% to about 6%, depending on the intended use andformulation ingredients (where “about” is ±10% of the indicated value).In certain embodiments, the antimicrobial cationic peptide is anindolicidin or an analog or derivative thereof in any one of theaforementioned compositions. In preferred embodiments, the antimicrobialcationic peptide is a peptide of up to 35 amino acids, comprising one ofthe following sequences: 11B7CN, 11B32CN, 11B36CN, 11E3CN, 11F4CN,11F5CN, 11F12CN, 11F17CN, 11F50CN, 11F56CN, 11F63CN, 11F64CN, 11F66CN,11F67CN, 11F68CN, 11F93CN, 11G27CN, 11J02CN, 11J02ACN, 11J30CN, 11J36CN,11J58CN, 11J67CN, 11J68CN, Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, orNt-glucosyl-11J38CN, which may be used in any one of the compositionsdescribed herein.

An antimicrobial cationic peptide of the present invention may be ananalog or derivative thereof. As used herein, the terms “derivative” and“analog” when referring to an antimicrobial cationic peptide,polypeptide, or fusion protein, refer to any antimicrobial cationicpeptide, polypeptide, or fusion protein that retain essentially the same(at least 50%, and preferably greater than 70, 80, or 90%) or enhancedbiological function or activity as such natural peptide, as noted above.The biological function or activity of such analogs and derivatives canbe determined using standard methods (e.g., antimicrobial,anti-inflammatory, DNA and/or protein synthesis inhibitor), such as withthe assays described herein. For example, an analog or derivative may bea proprotein that can be activated by cleavage to produce an activeantimicrobial cationic peptide. Alternatively, a cationic peptide analogor derivative thereof can be identified by the ability to specificallybind anti-cationic peptide antibodies.

A cationic peptide analog or derivative may have, for example, one ormore deletion, insertion, or modification of any amino acid residue,including the N- or C-terminal amino acids. Within the scope of thisinvention are modified antimicrobial cationic peptides, such as, forexample, peptides having an acetylated, acylated, acryloylated,alkylated, glycosylated (e.g., glucosylated), PEGylated, myristylated,and the like N-terminal amino acid modification; having an esterified,amidated, homoserine/homoserine lactone, or caprolactam C-terminal aminoacid modification; or having a polyalkylene glycol (e.g., polyethyleneglycol) conjugated to any free amino group. A preferred modification ofthe C-terminal amino acid is amidation.

An analog or derivative may also be an antimicrobial cationic peptidefusion protein. Fusion proteins, or chimeras, include fusions of one ormore antimicrobial cationic peptides, and fusions of cationic peptideswith non-cationic peptides. Additionally, the peptide may be modified toform a polymer-modified peptide. The peptides may also be labeled, suchas with a radioactive label, a fluorescent label, a mass spectrometrytag, biotin, and the like.

Another example of an analog or derivative includes an antimicrobialcationic peptide that has one or more conservative amino acidsubstitutions, as compared with the amino acid sequence of a naturallyoccurring cationic peptide. Among the common amino acids, a“conservative amino acid substitution” is illustrated, for example, by asubstitution among amino acids within each of the following groups: (1)glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine,tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate andglutamate, (5) glutamine and asparagine, and (6) lysine, arginine andhistidine, or a combination thereof. Furthermore, an analog orderivative of a cationic peptide may include, for example, non-proteinamino acids, such as precursors of normal amino acids (e.g., homoserineand diaminopimelate), intermediates in catabolic pathways (e.g.,pipecolic acid and D-enantiomers of normal amino acids), and amino acidanalogs (e.g., azetidine-2-carboxylic acid and canavanine).

The amino acid designations are herein set forth as either the standardone- or three-letter code. Unless otherwise indicated, a named aminoacid refers to the L-enantiomer. Polar amino acids include asparagine(Asp or N) and glutamine (Gln or Q); as well as basic amino acids suchas arginine (Arg or R), lysine (Lys or K), histidine (His or H), andderivatives thereof; and acidic amino acids such as aspartic acid (Aspor D) and glutamic acid (Glu or E), and derivatives thereof. Hydrophobicamino acids include tryptophan (Trp or W), phenylalanine (Phe or F),isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine(Val or V), and derivatives thereof; as well as other non-polar aminoacids such as glycine (Gly or G), alanine (Ala or A), proline (Pro orP), and derivatives thereof. Amino acids of intermediate polarityinclude serine (Ser or S), threonine (Thr or T), tyrosine (Tyr or Y),cysteine (Cys or C), and derivatives thereof. A capital letter indicatesan L-enantiomer amino acid; a small letter indicates a D-enantiomeramino acid. For example, some modified amino acids may include2,3-diamino butyric acid, 3- or 4-mercaptoproline derivatives,N⁵-acetyl-N⁵-hydroxy-L-ornitine, and α-N-hydroxyamino acids. Anantimicrobial cationic peptide analog or derivative thereof may includeany one or combination of the above-noted alterations to the naturalpeptide, or any other modification known in the art.

The indolicidins or analogs or derivatives thereof of the presentinvention may be used individually, or may be used in combination withone or more different indolicidins or analogs or derivatives thereof,with one or more antimicrobial cationic peptides, and one or moreconventional antimicrobial agents, as described herein. Thus,synergistic combinations of an antimicrobial cationic peptide and anantimicrobial agent may permit a reduction in the dosage of one or bothagents in order to achieve a similar or improved therapeutic effect.This would allow the use of smaller doses and, therefore, would decreasethe potential incidence of toxicity (e.g., from aminoglycosides) andlowering costs of expensive antimicrobials (e.g., vancomycin).Concurrent or sequential administration of an antimicrobial cationicpeptide formulation and an antimicrobial agent composition is expectedto provide more effective treatment of infections caused by a variety ofmicroorganisms (e.g., bacteria, viruses, fungi, and parasites). Inparticular, successful treatment or prevention of infectious disease canbe achieved by using the antimicrobial cationic peptides andantimicrobial agents at doses below what is normally a therapeuticallyeffective dose when these antimicrobials are used individually.Alternatively, the antibiotic agent and antimicrobial cationic peptideformulation can be administered using a normally effective therapeuticdose for each antimicrobial, but wherein the combination of the twoagents provides even more potent effects.

As noted above, the preferred antimicrobial cationic peptides may beused in a synergistic combination with other known antimicrobial agents.Antibacterial agents include, but are not limited to, penicillins,cephalosporins, carbacephems, cephamycins, carbapenems, monobactams,aminoglycosides, glycopeptides, quinolones, tetracyclines, macrolides,and fluoroquinolones. Examples of antibiotic agents include, but are notlimited to, Penicillin G (CAS Registry No.: 61-33-6); Methicillin (CASRegistry No.: 61-32-5); Nafcillin (CAS Registry No.: 147-52-4);Oxacillin (CAS Registry No.: 66-79-5); Cloxacillin (CAS Registry No.:61-72-3); Dicloxacillin (CAS Registry No.: 3116-76-5); Ampicillin (CASRegistry No.: 69-53-4); Amoxicillin (CAS Registry No.: 26787-78-0);Ticarcillin (CAS Registry No.: 34787-01-4); Carbenicillin (CAS RegistryNo.: 4697-36-3); Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin(CAS Registry No.: 37091-66-0); Piperacillin (CAS Registry No.:61477-96-1); Imipenem (CAS Registry No.: 74431-23-5); Aztreonam (CASRegistry No.: 78110-38-0); Cephalothin (CAS Registry No.: 153-61-7);Cefazolin (CAS Registry No.: 25953-19-9); Cefaclor (CAS Registry No.:70356-03-5); Cefamandole formate sodium (CAS Registry No.: 42540-40-9);Cefoxitin (CAS Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.:55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4); Cefmetazole (CASRegistry No.: 56796-20-4); Cefotetan (CAS Registry No.: 69712-56-7);Cefprozil (CAS Registry No.: 92665-29-7); Lincomycin (CAS Registry No.:154-21-2); Linezolid (CAS Registry No.: 165800-03-3); Loracarbef (CASRegistry No.: 121961-22-6); Cefetamet (CAS Registry No.: 65052-63-3);Cefoperazone (CAS Registry No.: 62893-19-0); Cefotaxime (CAS RegistryNo.: 63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0);Ceftriaxone (CAS Registry No.: 73384-59-5); Ceftazidime (CAS RegistryNo.: 72558-82-8); Cefepime (CAS Registry No.: 88040-23-7); Cefixime (CASRegistry No.: 79350-37-1); Cefpodoxime (CAS Registry No.: 80210-62-4);Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No.:79660-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin(CAS Registry No.: 70458-96-7); Ciprofloxacin (CAS Registry No.:85721-33-1); Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CASRegistry No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7);Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No.:564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CASRegistry No.: 60-54-8); Amikacin (CAS Registry No.: 37517-28-5);Gentamicin (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry No.:8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CASRegistry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1);Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CASRegistry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8);Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethylsuccinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate(CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS RegistryNo.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643-22-1);Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.:61036-64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin(CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.:738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin(CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1);Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS RegistryNo.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin(CAS Registry No.: 80214-83-1); Co-amoxiclavuanate; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives.

The antimicrobial cationic peptide may also be used in combination withanti-fungal agents. Exemplary anti-fungal agents include, but are notlimited to, terbinafine hydrochloride, nystatin, amphotericin B,griseofulvin, ketoconazole, miconazole nitrate, flucytosine,fluconazole, itraconazole, clotrimazole, benzoic acid, salicylic acid,and selenium sulfide.

The antimicrobial cationic peptide may also be used in combination withanti-viral agents. Exemplary anti-viral agents include, but are notlimited to, amantadine hydrochloride, rimantadin, acyclovir,famciclovir, foscarnet, ganciclovir sodium, idoxuridine, ribavirin,sorivudine, trifluridine, valacyclovir, vidarabin, didanosine,stavudine, zalcitabine, zidovudine, interferon alpha, and edoxudine.

The antimicrobial cationic peptide may also be used in combination withanti-parasitic agents. Exemplary anti-parasitic agents include, but arenot limited to, pirethrins/piperonyl butoxide, permethrin, iodoquinol,metronidazole, diethylcarbamazine citrate, piperazine, pyrantel pamoate,mebendazole, thiabendazole, praziquantel, albendazole, proguanil,quinidine gluconate injection, quinine sulfate, chloroquine phosphate,mefloquine hydrochloride, primaquine phosphate, atovaquone,co-trimoxazole (sulfamethoxazole/trimethoprim), and pentamidineisethionate.

In another aspect, antibodies may be generated to a specificantimicrobial cationic peptide and analogue or derivative thereof usingmultiple antigenic peptides (MAPs) that contain approximately eightcopies of the peptide linked to a small non-immunogenic peptidyl core toform an immunogen (see, in general, Antibodies: A Laboratory Manual,Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press, 1988).Alternatively, the target peptide may be conjugated to bovine serumalbumin (BSA), ovalbumin, or another suitable conjugate. The MAP orpeptide conjugate is injected subcutaneously into rabbits or into miceor other rodents, where they may have sufficiently long half-lives tofacilitate antibody production. After twelve weeks, blood samples aretaken and serum is separated for testing in an ELISA assay against theoriginal peptide, with a positive result indicating the presence ofantibodies specific to the target peptide. This serum can then be storedand used in ELISA assays to specifically measure the amount of thespecific antimicrobial cationic peptide and/or analog or derivativethereof. Alternatively, other standard methods of antibody productionmay be employed, for example generation of monoclonal antibodies.

Within the context of the present invention, antibodies are understoodto include, inter alia, monoclonal antibodies, polyclonal antibodies,anti-idiotypic antibodies, and antibody fragments (e.g., Fab, andF(ab′)₂, Fv variable regions, or complementarity determining regions).Antibodies are generally accepted as specific against cationic peptides,such as indolicidin and analogs or derivatives thereof, if they bindwith a K_(d) of greater than or equal to 10⁻⁷ M, preferably greater thanof equal to 10⁻⁸ M, and more preferably greater than of equal to 10⁻⁹ M.The affinity of a monoclonal antibody or binding partner may be readilydetermined by one of ordinary skill in the art (see, e.g., Scatchard,Ann. N.Y. Acad. Sci. 51:660-672, 1949). Once suitable antibodies havebeen identified, they may be isolated or purified by many techniqueswell known to those of ordinary skill in the art.

Monoclonal antibodies may also be readily generated from hybridoma celllines using conventional techniques (see U.S. Pat. Nos. RE 32,011,4,902,614, 4,543,439, and 4,411,993; see also Antibodies: A LaboratoryManual, 1988). Briefly, within one embodiment, a subject animal, such asa rat or mouse, is injected with a peptide of choice. The peptide isgenerally administered in an emulsion with an adjuvant, such as Freund'scomplete or incomplete adjuvant, which is intended to increase theimmune response. The animal is generally boosted at least once prior toharvest of the spleen and/or lymph nodes and immortalization of thosecells. Various immortalization techniques, such as mediated byEpstein-Barr virus or fusion to produce a hybridoma, may be used. In apreferred embodiment, immortalization occurs by fusion with a suitablemyeloma cell line to create a hybridoma that secretes monoclonalantibody. Suitable myeloma lines include, for example, NS-1 (ATCC No.TIB 18), and P3X63-Ag 8.653 (ATCC No. CRL 1580). The preferred fusionpartners do not express endogenous antibody genes. After about sevendays, the hybridomas may be screened for the presence of antibodies thatare reactive against an antimicrobial cationic peptide and analog orderivative thereof. A wide variety of assays may be utilized (seeAntibodies: A Laboratory Manual, 1988).

Other techniques known in the art may be utilized to constructmonoclonal antibodies (see Huse et al., Science 246:1275-1281, 1989;Sastry et al., Proc. Natl. Acad. Sci. USA 86:5728-5732, 1989;Alting-Mees et al., Strategies in Molecular Biology 3:1-9, 1990;describing recombinant techniques). These techniques include cloningheavy and light chain immunoglobulin cDNA in suitable vectors, such asλImmunoZap(H) and λ ImmunoZap(L). These recombinants may be screenedindividually or co-expressed to form Fab fragments or antibodies (seeHuse et al., supra; Sastry et al., supra). Positive plaques maysubsequently be converted into non-lytic plasmids to allow high-levelexpression of monoclonal antibody fragments in a host, such as E. coli.

Similarly, portions or fragments of antibodies, such as Fab and Fvfragments, may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to yield isolated variableregions of an antibody. Within one embodiment, the genes that encode thevariable region from a hybridoma producing a monoclonal antibody ofinterest are amplified using nucleotide primers for the variable region.In addition, techniques may be utilized to change a “murine” antibody toa “human” antibody, without altering the binding specificity of theantibody to the antimicrobial cationic peptide and analog or derivativethereof.

B. Nucleic Acids Encoding Antimicrobial Cationic Peptides

Nucleic acid molecules encoding cationic peptides may be isolated fromnatural sources, may be obtained by automated synthesis of nucleic acidmolecules, or may be obtained by using the polymerase chain reaction(PCR) with oligonucleotide primers having nucleotide sequences that arebased upon known nucleotide sequences of antimicrobial cationic peptidegenes. In the latter approach, a cationic peptide gene is synthesizedusing mutually priming oligonucleotides (see, for example, Ausubel etal. (eds.), Short Protocols in Molecular Biology, 3^(rd) Edition, pages8-8 to 8-9, John Wiley & Sons, 1995, herein after referred to as“Ausubel (1995)”). Established techniques using the polymerase chainreaction provide the ability to synthesize DNA molecules of at least twokilobases in length (Adang et al., Plant Molec. Biol. 21:1131, 1993;Bambot et al., PCR Methods and Applications 2:266, 1993; Dillon et al.,“Use of the Polymerase Chain Reaction for the Rapid Construction ofSynthetic Genes,” in Methods in Molecular Biology, Vol. 15: PCRProtocols: Current Methods and Applications, White (ed.), pages 263-268,Humana Press, Inc., 1993; Holowachuk et al., PCR Methods Appl. 4:299,1995).

As noted above, the present invention contemplates analogs orderivatives of natural cationic peptides, which analogs or derivativesmay be recombinantly produced by the presently described methods.Nucleotide sequences encoding conservative amino acid analogs orderivatives can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like (see Ausubel, 1995, at page 8-10through page 8-22; and McPherson (ed.), Directed Mutagenesis: APractical Approach, IRL Press, 1991).

Although one objective in constructing a cationic peptide variant may beto improve its activity, it may also be desirable to alter the aminoacid sequence of a naturally occurring cationic peptide to enhance itsproduction in a recombinant host cell. The presence of a particularcodon may have an adverse effect on expression in a particular host;therefore, a DNA sequence encoding the desired cationic peptide isoptimized for a particular host system, such as prokaryotic oreukaryotic cells. For example, a nucleotide sequence encoding a radishcationic peptide may include a codon that is commonly found in radish,but is rare for E. coli. The presence of a rare codon may have anadverse effect on protein levels when the radish cationic peptide isexpressed in recombinant E. coli. Methods for altering nucleotidesequences to alleviate the codon usage problem are well known to thoseof skill in the art (see, e.g., Kane, Curr. Opin. Biotechnol. 6:494,1995; Makrides, Microbiol. Rev. 60:512, 1996; and Brown (Ed.), MolecularBiology LabFax, BIOS Scientific Publishers, Ltd., 1991, which provides aCodon Usage Table at page 245 through page 253).

Peptides may be synthesized by recombinant techniques (see e.g., U.S.Pat. No. 5,593,866) and a variety of host systems are suitable forproduction of the cationic peptides and analogues or derivativesthereof, including bacteria (e.g., E. coli), yeast (e.g., Saccharomycescerevisiae), insect (e.g., Sf9), and mammalian cells (e.g., CHO, COS-7).Many expression vectors have been developed and are available for eachof these hosts. Generally, vectors that are functional in bacteria areused in this invention. However, at times, it may be preferable to havevectors that are functional in other hosts. Vectors and procedures forcloning and expression in E. coli are discussed herein and, for example,in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1987) and in Ausubelet al., 1995.

A DNA sequence encoding a cationic peptide is introduced into anexpression vector appropriate for the host. In preferred embodiments,the gene is cloned into a vector to create a fusion protein. The fusionpartner is chosen to contain an anionic region, such that a bacterialhost is protected from the toxic effect of the peptide. This protectiveregion effectively neutralizes the antimicrobial effects of the peptideand also may prevent peptide degradation by host proteases. The fusionpartner (carrier protein) of the invention may further function totransport the fusion peptide to inclusion bodies, the periplasm, theouter membrane, or the extracellular environment. Carrier proteinssuitable in the context of this invention specifically include, but arenot limited to, glutathione-S-transferase (GST), protein A fromStaphylococcus aureus, two synthetic IgG-binding domains (ZZ) of proteinA, outer membrane protein F, β-galactosidase (lacZ), and variousproducts of bacteriophage λ and bacteriophage T7. From the teachingsprovided herein, it is apparent that other proteins may be used ascarriers. Furthermore, the entire carrier protein need not be used, aslong as the protective anionic region is present. To facilitateisolation of the peptide sequence, amino acids susceptible to chemicalcleavage (e.g., CNBr) or enzymatic cleavage (e.g., V8 protease, trypsin)are used to bridge the peptide and fusion partner. For expression in E.coli, the fusion partner is preferably a normal intracellular proteinthat directs expression toward inclusion body formation. In such a case,following cleavage to release the final product, there is no requirementfor renaturation of the peptide. In the present invention, the DNAcassette, comprising fusion partner and peptide gene, may be insertedinto an expression vector, which can be a plasmid, virus or othervehicle known in the art. Preferably, the expression vector is a plasmidthat contains an inducible or constitutive promoter to facilitate theefficient transcription of the inserted DNA sequence in the host.Transformation of the host cell with the recombinant DNA may be carriedout by Ca⁺⁺-mediated techniques, by electroporation, or other methodswell known to those skilled in the art.

Briefly, a DNA fragment encoding a peptide is derived from an existingcDNA or genomic clone or synthesized. A convenient method isamplification of the gene from a single-stranded template. The templateis generally the product of an automated oligonucleotide synthesis.Amplification primers are derived from the 5′ and 3′ ends of thetemplate and typically incorporate restriction sites chosen with regardto the cloning site of the vector. If necessary, translationalinitiation and termination codons can be engineered into the primersequences. The sequence encoding the protein may be codon-optimized forexpression in the particular host. Thus, for example, if the analoguefusion protein is expressed in bacteria, codons are optimized forbacterial usage. Codon optimization is accomplished by automatedsynthesis of the entire gene or gene region, ligation of multipleoligonucleotides, mutagenesis of the native sequence, or othertechniques known to those in the art.

Within a preferred embodiment, the vector is capable of replication inbacterial cells. Thus, the vector may contain a bacterial origin ofreplication. Preferred bacterial origins of replication include f1-oriand col E1 ori, especially the ori derived from pUC plasmids. Low copynumber vectors (e.g., pPD100) may also be used, especially when theproduct is deleterious to the host. The plasmids also preferably includeat least one selectable marker that is functional in the host. Aselectable marker gene confers a phenotype on the host that allowstransformed cells to be identified and/or selectively grown. Suitableselectable marker genes for bacterial hosts include the chloroamphenicolresistance gene (Cm^(r)), ampicillin resistance gene (Amp^(r)),tetracycline resistance gene (Tc^(r)) kanamycin resistance gene(Kan^(r)), and others known in the art. To function in selection, somemarkers may require a complementary deficiency in the host. The vectormay also contain a gene coding for a repressor protein, which is capableof repressing the transcription of a promoter that contains a repressorbinding site. Altering the physiological conditions of the cell candepress the promoter. For example, a molecule may be added thatcompetitively binds the repressor, or the temperature of the growthmedia may be altered. Repressor proteins include, but are not limited tothe E. coli lacI repressor (responsive to induction by IPTG), thetemperature sensitive λc1857 repressor, and the like.

At minimum, the expression vector should contain a promoter sequence.However, other regulatory sequences may also be included. Such sequencesinclude an enhancer, ribosome binding site, transcription terminationsignal sequence, secretion signal sequence, origin of replication,selectable marker, and the like. The regulatory sequences are operablylinked with one another to allow transcription and subsequenttranslation. In preferred aspects, the plasmids used herein forexpression include a promoter designed for expression of the proteins inbacteria. Suitable promoters, including both constitutive and induciblepromoters, are widely available and are well known in the art. Commonlyused promoters for expression in bacteria include promoters from T7, T3,T5, and SP6 phages, and the trp, lpp, and lac operons. Hybrid promoters(see, U.S. Pat. No. 4,551,433), such as tac and trc, may also be used.Examples of plasmids for expression in bacteria include the pETexpression vectors pET3a, pET 11a, pET 12a-c, and pET 15b (see U.S. Pat.No. 4,952,496; available from Novagen, Madison, Wis.). Low copy numbervectors (e.g., pPD100) can be used for efficient overproduction ofpeptides deleterious to the E. coli host (Dersch et al., FEMS Microbiol.Lett. 123: 19, 1994). Bacterial hosts for the T7 expression vectors maycontain chromosomal copies of DNA encoding T7 RNA polymerase operablylinked to an inducible promoter (e.g., lacUV promoter; see, U.S. Pat.No. 4,952,496), such as found in the E. coli strains HMS174(DE3)pLysS,BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3). T7 RNA polymerase can also bepresent on plasmids compatible with the T7 expression vector. Thepolymerase may be under control of a lambda promoter and repressor(e.g., pGP1-2; Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82:1074,1985).

In some aspects, the sequence of nucleotides encoding the peptide alsoencodes a secretion signal, such that the resulting peptide issynthesized as a precursor protein (i.e., proprotein), which issubsequently processed and secreted. The resulting secreted peptide orfusion protein may be recovered from the periplasmic space or thefermentation medium. Sequences of secretion signals suitable for use arewidely available and are well known (von Heijne, J. Mol. Biol.184:99-105, 1985).

The peptide product is isolated by standard techniques, such asaffinity, size exclusion, or ionic exchange chromatography, HPLC and thelike. An isolated peptide should preferably show a major band byCoomassie blue stain of SDS-PAGE, which is preferably at least 75%, 80%,90%, or 95% of the purified peptide, polypeptide, or fusion protein.

C. Testing Antimicrobial Cationic Peptides Analogs and Derivatives

Antimicrobial cationic peptides, and analogs or derivatives thereof, ofthe present invention are assessed, either alone or in combination withan antimicrobial agent or another analog, for their potential asantibiotic therapeutic agents using a series of assays. Preferably, allpeptides are initially assessed in vitro, the most promising candidatesare selected for further assessment in vivo, and then candidates areselected for pre-clinical studies. The in vitro assays includemeasurement of antibiotic activity, toxicity, solubility, pharmacology,secondary structure, liposome permeabilization and the like. In vivoassays include assessment of efficacy in animal models, antigenicity,toxicity, and the like. In general, in vitro assays are initiallyperformed, followed by in vivo assays.

Generally, cationic peptides are initially tested for (1) antimicrobialactivity in vitro; (2) in vitro toxicity to normal mammalian cells; and(3) in vivo toxicity in an animal model. Cationic peptides that havesome antimicrobial activity are preferred, although such activity maynot be necessary for enhancing the activity of an antibiotic agent.Also, for in vivo use, peptides should preferably demonstrate acceptabletoxicity profiles, as measured by standard procedures, where lowertoxicity is preferred. Additional assays may be performed to demonstratethat the peptide is not immunogenic and to examine antimicrobialactivity in vivo.

1. In Vitro Assays

Cationic peptides, including indolicidin analogues, are assayed by, forexample, an agarose dilution MIC assay, a broth dilution, time-killassay, or equivalent methods. Antimicrobial activity is measured asinhibition of growth or killing of a microorganism (e.g., bacteria,fungi).

Briefly, a candidate antimicrobial cationic peptide in Mueller Hintonbroth supplemented with calcium and magnesium is mixed with moltenagarose. Other broths and agars may be used as long as the peptide canfreely diffuse through the medium. The agarose is poured into petridishes or wells, allowed to solidify, and a test strain is applied tothe agarose plate. The test strain is chosen, in part, on the intendedapplication of the peptide. Thus, by way of example, if an indolicidinor analog or derivative thereof with activity against S. aureus isdesired, a S. aureus strain is used. It may be desirable to assay thecandidate antimicrobial cationic peptide on several strains and/or onclinical isolates of the test species. Plates are incubated overnightand inspected visually for bacterial growth. A minimum inhibitoryconcentration (MIC) of a cationic peptide is the lowest concentration ofpeptide that completely inhibits growth of the organism. Peptides thatexhibit good activity against the test strain, or group of strains,typically having an MIC of less than or equal to 16 μg/ml are selectedfor further testing. Preferred antimicrobial cationic peptides oranalogs or derivatives thereof may be microbicidal or microbistatic.

Alternatively, time kill curves can be used to determine the differencein growth (e.g., bacterial colony counts) over a set time period,typically 24 hours. Briefly, a suspension of organisms at a knownconcentration is prepared and a candidate peptide is added. Aliquots ofthe suspension are removed at set times, diluted, plated on medium,incubated, and counted. MIC is measured as the lowest concentration ofpeptide that completely inhibits growth of the organism and, in general,lower MIC values are preferred.

Solubility of the peptide in a solvent, broth, or co-solvent system asdescribed herein is an additional parameter that may be examined.Several different assays may be used, such as appearance in buffer.Briefly, a candidate antimicrobial cationic peptide or analog orderivative thereof may be contacted with a solvent, broth, or co-solventsystem, and the appearance evaluated according to a scale that rangesfrom (a) clear, no precipitate, (b) light, diffuse precipitate, to (c)cloudy, heavy precipitate. In general, less precipitate is moredesirable, but some precipitate may be acceptable. To assess the levelof solubility, for example, a person having ordinary skill in the artmay inspect the combination visually, or a variety of spectrophotometrictechniques may be used, such as by U.V. or visible light absorbance atthe appropriate wavelength.

Additional in vitro assays may be carried out to assess the potential ofa candidate peptide or analog or derivative thereof as a therapeuticagent. Such assays include peptide solubility in formulations,pharmacology and stability in blood or plasma, serum protein binding,analysis of secondary structure (e.g., by circular dichroism), liposomepermeabilization, and bacterial membrane permeabilization. In general, apreferred embodiment includes a candidate peptide analog or derivativethereof that is soluble, is active in biological fluids, is stable, andhas generally greater antimicrobial activity than the natural peptide(e.g., indolicidin).

2. In Vivo Assays

Peptides and analogs or derivative thereof, selected on the basis of theresults from the in vitro assays can be further tested in vivo forefficacy, stability, and the like. A variety of methods and animalmodels are available to assess the antimicrobial activity of selectedcandidate peptides and analogs or derivative thereof in vivo for theirability to ameliorate microbial infections. Within these assays, apeptide is useful as a therapeutic if inhibition of microbial growthcompared to inhibition with the vehicle alone is statisticallysignificant. This measurement can be made directly from culturesisolated from body fluids or sites, or indirectly by assessing survivalrates of infected animals.

For assessment of antibacterial activity of candidate peptide analogsand derivatives as compared to natural peptides, several animal modelsare available, such as acute infection models including those in which(a) normal mice receive a lethal dose of microorganisms, (b) neutropenicmice receive a lethal dose of microorganisms, or (c) rabbits receive aninoculum of microorganisms in the heart, and chronic infection models.The model selected will depend, in part, on the intended clinicalindication of the peptide and/or analog or derivative thereof.

By way of example and not limitation, a normal mouse model is used toinoculate mice, intraperitoneally (i.p.) or intravenously (i.v.), with alethal dose of bacteria. Typically, the dose is such that 90-100% ofanimals die within 2 days. The choice of a microorganism strain for thisassay depends, in part, upon the intended application of theantimicrobial cationic peptide or analog or derivative thereof, and inthe accompanying examples, assays are carried out with three differentStaphylococcus strains. Briefly, shortly before or after inoculationwith the microorganism of choice (generally within 60 minutes), peptidesor analogs or derivatives thereof in a suitable formulation buffer (asdescribed herein) is injected. Multiple injections of peptides may beadministered. Animals are observed for up to 8 days post-infection, andthe survival of animals is recorded. Successful treatment either rescuesanimals from death or delays death to a statistically significant level,as compared with non-treatment, control animals. Antimicrobial cationicpeptide analogs or derivatives thereof that show better efficacy thanthe natural peptides, such as indolicidins, are preferred.

Furthermore, for in vivo use, low immunogenicity is preferred. Tomeasure immunogenicity, peptides are injected into normal animals,generally rabbits. At various times after single or multiple injections,serum is obtained and tested for antibody reactivity to the peptide oranalog or derivative thereof. Antibodies to peptides or analogs orderivatives thereof may be identified by ELISA, immunoprecipitationassays, western blots, and other methods known in the art (seeAntibodies: A Laboratory Manual, 1988). In a preferred embodiment theantibody of interest has undetectable, or minimally detectable,reactivity with the candidate peptides or analogs or derivative thereof.In addition, pharmacokinetics of the candidate peptides or analogs orderivatives in animals and histopathology of animals treated with thepeptides may be determined.

Selection of antimicrobial cationic peptides and analogs or derivativesthereof as potential therapeutics is typically based on in vitro and invivo assay results. In general, peptides that exhibit lowimmunogenicity, good in vivo stability, and high efficacy at low doselevels are preferred candidate antimicrobial cationic peptides andanalogs or derivatives thereof.

D. Cationic Peptide Formulations and Therapeutic Methods

As noted above, the present invention provides methods for treating andpreventing infections by administering to a patient a therapeuticallyeffective amount of an antimicrobial cationic peptide, preferably anindolicidin or analog or derivative thereof, as described herein. Theantimicrobial cationic peptide is preferably part of a pharmaceuticalcomposition when used in the methods of the present invention. Thepharmaceutical composition will include at least one of apharmaceutically acceptable vehicle, carrier, diluent, or excipient, inaddition to one or more antimicrobial cationic peptide and, optionally,other components. Pharmaceutically acceptable excipients for therapeuticuse are well known in the pharmaceutical art, and are described hereinand, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro, ed., 18^(th) Edition, 1990) and in CRCHandbook of Food, Drug, and Cosmetic Excipients, CRC Press LLC (S. C.Smolinski, ed., 1992).

The therapeutic efficacy of an antimicrobial cationic peptidecomposition according to the present invention is based on a successfulclinical outcome and does not require 100% elimination of themicroorganisms involved in the infection. Achieving a level ofantimicrobial activity at the site of infection that allows the host tosurvive, resolve the infection, or eradicate the causative agent issufficient. When host defenses are maximally effective, such as in anotherwise healthy individual, only a minimal antimicrobial effect maysuffice. Thus, reducing the organism load by even one log (a factor of10) may permit the defenses of the host to control the infection. Inaddition, clinical therapeutic success may depend more on augmenting anearly bactericidal effect rather than on a long-term effect because thisallows time for activation of host defense mechanisms. This isespecially true for life-threatening infections (e.g., meningitis) andother serious chronic infections (e.g., infective endocarditis).

The formulations of the present invention, having an amount ofantimicrobial cationic peptide sufficient to treat or prevent aninfection are, for example, particularly suitable for topical (e.g.,creams, ointments, skin patches, eye drops, ear drops, shampoos)application or administration. Other typical routes of administrationinclude, without limitation, oral, parenteral, sublingual, bladderwash-out, vaginal, rectal, enteric, suppository, nasal, and inhalation.The term parenteral, as used herein, includes subcutaneous, intravenous,intramuscular, intraarterial, intraabdominal, intraperitoneal,intraarticular, intraocular or retrobulbar, intraaural, intrathecal,intracavitary, intracelial, intraspinal, intrapulmonary ortranspulmonary, intrasynovial, and intraurethral injection or infusiontechniques. The pharmaceutical compositions of the present invention areformulated so as to allow the antimicrobial cationic peptide containedtherein to be bioavailable upon administration of the composition to asubject. The level of peptide in serum and other tissues afteradministration can be monitored by various well-established techniques,such as bacterial, chromatographic or antibody based (e.g., ELISA)assays. Thus, in certain preferred embodiments, antimicrobial cationicpeptides and analogs and derivatives thereof, as described herein, areformulated for topical application to a target site on a subject in needthereof, such as an animal or a human.

The compositions may be administered to a subject as a single dosageunit (e.g., a tablet, capsule, or gel), and the compositions may beadministered as a plurality of dosage units (e.g., in aerosol form). Forexample, the antimicrobial cationic peptide formulations may besterilized and packaged in single-use, plastic laminated pouches orplastic tubes of dimensions selected to provide for routine, measureddispensing. In one example, the container may have dimensionsanticipated to dispense 0.5 ml of the antimicrobial cationic peptidecomposition (e.g., a gel form) to a limited area of the target surfaceon or in a subject to treat or prevent an infection. A typical target,for example, is in the immediate vicinity of the insertion site of anintravenous catheter, where the target surface usually has an area ofabout two square centimeters.

The antimicrobial cationic peptide composition may be provided invarious forms, depending on the amount and number of differentpharmaceutically acceptable excipients present. For example, the peptidecomposition may be in the form of a solid, a semi-solid, a liquid, alotion, a cream, an ointment, a cement, a paste, a gel, or an aerosol.In a preferred embodiment, the peptide formulation is in the form of agel. The pharmaceutically acceptable excipients suitable for use in thepeptide formulation compositions as described herein may include, forexample, a viscosity-increasing agent, a buffering agent, a solvent, ahumectant, a preservative, a chelating agent, an oleaginous compound, anemollient, an antioxidant, an adjuvant, and the like. The function ofeach of these excipients is not mutually exclusive within the context ofthe present invention. For example, glycerin may be used as a solvent oras a humectant or as a viscosity-increasing agent. In one preferredembodiment, the formulation is a composition comprising an antimicrobialcationic peptide, a viscosity-increasing agent, and a solvent, which isuseful, for example, at a target site for implanted or indwellingmedical devices, as described herein.

The pharmaceutically acceptable excipients noted above are known in theart, yet the unexpected result of the present invention is thatparticular combinations of excipients afford the antimicrobial cationicpeptides and analogs and derivatives thereof stability and prolongedactivity when stored at ambient temperature, even in the presence of analcoholic solvent. Solvents useful in the present compositions are wellknown in the art and include without limitation water, glycerin,propylene glycol, isopropanol, ethanol, and methanol. In someembodiments, the solvent is glycerin or propylene glycol, preferably ata concentration ranging from about 0.1% to about 20%, more preferablyabout 5% to about 15%, and most preferably about 9% to 11%. In otherembodiments, the solvent is water or ethanol, preferably at aconcentration up to about 99%, more preferably up to about 90%, and mostpreferably up to about 85%. (Unless otherwise indicated, all percentagesare on a w/w basis.) In yet other embodiments, the solvent is at leastone of water, glycerin, propylene glycol, isopropanol, ethanol, andmethanol, preferably is glycerin or propylene glycol and ethanol, morepreferably is glycerin and ethanol, and most preferably is glycerin andwater. One embodiment is a composition comprising an antimicrobialcationic peptide, a viscosity-increasing agent, a solvent, wherein thesolvent comprises at least one of water at a concentration up to 99%,glycerin at a concentration up to 20%, propylene glycol at aconcentration up to 20%, ethanol at a concentration up to 99%, andmethanol at a concentration up to 99%.

Another useful pharmaceutical excipient of the present invention is aviscosity-increasing agent. In certain embodiments, the antimicrobialcationic peptide compositions of the present invention include aviscosity-increasing agent, including without limitation dextran,polyvinylpyrrolidone, methylcellulose, carboxymethyl cellulose,hydroxyethyl cellulose, hydroxypropyl methylcellulose, and hydroxypropylcellulose, and combinations thereof. In preferred embodiments, theviscosity-increasing agent is hydroxyethyl cellulose or hydroxypropylmethylcellulose, preferably at a concentration ranging from about 0.5%to about 5%, more preferably from about 1% to about 3%, most preferablyfrom about 1.3% to about 1.7%. In yet other preferred embodiments, theantimicrobial cationic peptide compositions have a firstviscosity-increasing agent, such as hydroxyethyl cellulose,hydroxypropyl methylcellulose, dextran, or polyvinylpyrrolidone, and asecond viscosity-increasing agent such as hydroxyethyl cellulose,hydroxypropyl methylcellulose, dextran, or polyvinylpyrrolidone. Whenused as either a first or second viscosity-increasing agent, dextran andpolyvinylpyrrolidone are preferably used at a concentration ranging fromabout 0.1% to about 5% and more preferably from about 0.5% to about 1%.In one preferred embodiment, the first viscosity-increasing agent ishydroxyethyl cellulose at a concentration up to 3% and the secondviscosity-increasing agent is hydroxypropyl methylcellulose at aconcentration up to 3%. As is known in the art, the amount ofviscosity-increasing agent may be increased to shift the form of thecomposition from a liquid to a gel to a semi-solid form. Thus, theamount of a viscosity-increasing agent used in a formulation may bevaried depending on the intended use and location of administration ofthe peptide compositions provided herein.

In certain applications, it may be desirable to maintain the pH of theantimicrobial cationic peptide composition contemplated by the presentinvention within a physiologically acceptable range and within a rangethat optimizes the activity of the peptide or analog or derivativethereof. The antimicrobial cationic peptides of the present inventionfunction best in a composition that is neutral or somewhat acidic,although the peptides will still have antimicrobial andanti-inflammatory activity in a composition that is slightly basic(i.e., pH 8). Accordingly, a composition comprising an antimicrobialcationic peptide, a viscosity-increasing agent, and a solvent, mayfurther comprise a buffering agent. In certain embodiments, thebuffering agent comprises a monocarboxylate or a dicarboxylate, and morespecifically may be acetate, fumarate, lactate, malonate, succinate, ortartrate. Preferably, the antimicrobial cationic peptide compositionhaving the buffering agent has a pH ranging from about 3 to about 8, andmore preferably from about 3.5 to 7. In another preferred embodiment,the buffering agent is at a concentration ranging from about 1 mM toabout 200 mM, and more preferably from about 2 mM to about 20 mM, andmost preferably about 4 mM to about 6 mM.

Other optional pharmaceutically acceptable excipients are those thatmay, for example, aid in the administration of the formulation (e.g.,anti-irritant, polymer carrier, adjuvant) or aid in protecting theintegrity of the components of the formulation (e.g., anti-oxidants andpreservatives. Typically, a 1.0% antimicrobial cationic peptidecomposition may be stored at 2° C. to 8° C. In certain embodiments, thecomposition comprising an antimicrobial cationic peptide, aviscosity-increasing agent, and a solvent, may further comprise ahumectant, preferably sorbitol and the like, or a preservative,preferably benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben,propylparaben, and the like. In certain circumstances, the antimicrobialcationic peptide or analog or derivative thereof may itself function asa preservative of the final therapeutic composition. For example, apreservative is optional in the gel formulations described hereinbecause the gels may be sterilized by autoclaving and, furthermore, showthe surprising quality of releasing (i.e., making bioavailable) theantimicrobial cationic peptide at a more optimal rate than otherformulations, such as a cream. In addition, particular embodiments mayhave in a single formulation a humectant, a preservative, and abuffering agent, or combinations thereof. Therefore, a preferredembodiment is a composition comprising an antimicrobial cationicpeptide, a viscosity-increasing agent, a solvent, a humectant, and abuffering agent. Another preferred embodiment is a compositioncomprising an antimicrobial cationic peptide, a viscosity-increasingagent, a buffering agent, and a solvent. In yet another preferredembodiment, the composition comprises an antimicrobial cationic peptide,a buffering agent, and a solvent. Each of the above formulations may beused to treat or prevent infection or to reduce the microflora at atarget site, such as a catheter insertion site on a subject (i.e.,animal or human).

In yet other embodiments, the composition is in the form of an ointmentcomprising an antimicrobial cationic peptide (preferably in an amountsufficient to treat or prevent an infection) and an oleaginous compound.For example, oleaginous compound may be petrolatum. In one embodiment,the oleaginous compound is present at a concentration ranging from about50% to about 100%, more preferably from about 70% to about 100%, evenmore preferably from about 80% to about 100%, and most preferably fromabout 95% to about 100%. In certain other embodiments, the ointmentcomposition may further comprise at least one emollient. The emollientsmay be present at a concentration ranging from about 1% to about 40%,more preferably from about 5% to about 30%, and more preferably fromabout 5% to about 10%. In certain preferred embodiments, the emollientmay be mineral oil, cetostearyl alcohol, glyceryl stearate, and acombination thereof. In another aspect the composition is in the form ofa semi-solid emulsion (e.g., a cream) comprising an antimicrobialcationic peptide (preferably in an amount sufficient to treat or preventan infection), a solvent, a buffering agent, at least one emollient, andat least one emulsifier. In a preferred embodiment, the semi-solidemulsion or cream further comprises at least one of a humectant (e.g.,sorbitol and/or glycerin), an oleaginous compound (e.g., petrolatum), aviscosity increasing agent (e.g., dextran, polyvinylpyrrolidone,hydroxyethyl cellulose, and/or hydroxypropyl methylcellulose), ananti-oxidant (e.g., butylated hydroxytoluene and preferably at aconcentration ranging from about 0.01% to about 0.1%), a preservative(e.g., benzoic acid, benzyl alcohol, phenoxyethanol, methylparaben,propylparaben, or a combination thereof), or a combination thereof. Incertain preferred embodiments, the emollient may be one or more ofstearyl alcohol, cetyl alcohol, and mineral oil. In certain otherpreferred embodiments, the emulsifiers may be one or more of stearylalcohol, cetyl alcohol, polyoxyethylene 40 stearate, and glycerylmonostearate. In a preferred embodiment, the emulsifier is present at aconcentration ranging from about 1% to about 20%, more preferably fromabout 5% to about 10, and most preferably from about 1% to about 1.5%.As noted above, the function of each of these emulsifiers and emollientsis not mutually exclusive in that an emollient may function as anemulsifier and the emulsifier may function as an emollient, depending onthe particular formulation, as is known in the art and is describedherein. In certain preferred embodiments the solvent comprises water andthe like, and the buffering agent comprises a monocarboxylate ordicarboxylate and the like, as described herein.

A subject suitable for treatment with an antimicrobial peptideformulation may be identified by well-established indicators of risk fordeveloping a disease or well-established hallmarks of an existingdisease. For example, indicators of an infection include fever, pus,microorganism positive cultures, inflammation, and the like. Infectionsthat may be treated with antimicrobial cationic peptides provided by thepresent invention include without limitation those caused by or due tomicroorganisms, whether the infection is primary, secondary,opportunistic, or the like. Examples of microorganisms include bacteria(e.g., Gram-positive, Gram-negative), fungi, (e.g., yeast and molds),parasites (e.g., protozoans, nematodes, cestodes and trematodes),viruses (e.g., HIV, HSV, VSV), algae, and prions. Specific organisms inthese classes are well known (see, for example, Davis et al.,Microbiology, 3^(rd) edition, Harper & Row, 1980; and Stanier et al.,The Microbial World, 5^(th) edition, Prentice Hall, 1986). Infectionsinclude, but are not limited to, toxic shock syndrome, diphtheria,cholera, typhus, meningitis, whooping cough, botulism, tetanus, pyogenicinfections, sinusitis, pneumonia, gingivitis, mucitis, folliculitis,cellulitis, acne and acne vulgaris, impetigo, osteomyelitis,endocarditis, ulcers, burns, dysentery, urinary tract infections,gastroenteritis, anthrax, Lyme disease, syphilis, rubella, septicemia,and plague; as well as primary, secondary, and opportunistic infectionsassociated with, for example, trauma, surgery, endotracheal intubation,tracheostomy, and cystic fibrosis.

A subject may have other clinical indications treatable or preventablewith the compositions and methods of the present invention, whichinclude without limitation those associated with implantable,indwelling, or similar medical devices, such as intravascular catheters(e.g., intravenous and intra-arterial), right heart flow-directedcatheters, Hickman catheters, arteriovenous fistulae, catheters used inhemodialysis and peritoneal dialysis (e.g., silastic, central venous,Tenckhoff, and teflon catheters), vascular access ports, indwellingurinary catheters, urinary catheters, silicone catheters, ventricularcatheters, synthetic vascular prostheses (e.g., aortofemoral andfemoropopliteal), prosthetic heart valves, prosthetic joints, orthopedicimplants, penile implants, shunts (e.g., Scribner, Torkildsen, centralnervous system, portasystemic, ventricular, ventriculoperitoneal),intrauterine devices, tampons, contact lenses, dental implants, ureteralstents, pacemakers, implantable defibrillators, tubing, cannulas,probes, blood monitoring devices, needles, and the like. As used herein,“medical device” refers to any device for use in a subject, such as ananimal or human.

By way of background, each year over 5 million central venous catheter(CVC) units are sold in the U.S., and it is estimated that 250,000patients in the U.S. develop bloodstream infections related to CVCs eachyear. Infections associated with CVCs of various types account for80-90% of all vascular catheter-related bloodstream infections (seeMaki, D. G., Infections Caused by Intravascular Devices Used forInfusion Therapy: Pathogenesis, Prevention, and Management, In:Infections Associated with Indwelling Medical Devices, 2^(nd) ed., A. L.Bisno and F. A. Waldvogel (eds.), American Society for Microbiology,Washington, D.C., 1994). Prospective studies of short-term, non-cuffed,single or multilumen catheters inserted percutaneously into thesubclavian or internal jugular vein have shown that rates ofcatheter-related septicemia range from 3-5%, with rates of 7-10% in somehospitals (Maki, 1994). Some of the organisms most commonly found to becausing these infections are Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecium, Escherichia coli, Enterobactercloacae, Pseudomonas aeruginosa, and Candida albicans, which account for95% of reported infections. Therefore, a subject having a catheter orscheduled to have a catheter inserted would be particularly benefited bythe antimicrobial cationic peptide compositions and methods of thepresent invention.

The primary strategies to prevent catheter-related infection are barrierprecautions and the use of specialized i.v. teams. Vigorous hand washingand sterile gloves is highly recommended prior to catheter insertion.Other barrier precautions include, for example, a long-sleeved andsterile surgical gown, a mask, a cap, and a large sterile drape. Thisbarrier methodology used in conjunction with specialized i.v. teams hasbeen found to be effective at reducing rates of, for example,catheter-related infection. However, even when these precautions aretaken, the longer any type of intravascular device remains in place, thehigher the cumulative risk of device-related infection (particularlysepticemia). For example, the length of time that non-cuffed CVCs areallowed to remain in place in Intensive Care Unit (ICU) patients isarbitrarily limited to 3 to 7 days in many medical centers. When thecontinued use of a short-term CVC for more than 4 days is consideredessential in an ICU patient, the clinician has three options: (i) leavethe catheter in place, accepting that the risk of infection willincrease after 4 days; (ii) replace the old catheter over a guidewire atthe original insertion site; or (iii) remove the catheter and place anew catheter at a new site, thereby gaining another 4 days of low risk.None of these options are optimal for the clinician or the patient.

An advantage of the present invention is that antisepsis of a targetsite for device insertion with an indolicidin or analog or derivativethereof, and formulated as described herein, may be achieved prior todevice insertion and/or after device insertion and in combination withbarrier precautions, which will reduce the risk of device-associated andother infections. Therefore, the compositions and methods of the presentinvention are specifically useful, for example, for cutaneous antisepsisto treat or prevent localized infection (e.g., after trauma, surgery, orother medical procedure), and to prevent medical device-relatedsepticemia. Furthermore, as noted above, one benefit of usingantimicrobial cationic peptides is the reduction of the risk forselecting antimicrobial-resistant microorganisms.

Uses of antimicrobial cationic peptide formulations of the presentinvention encompass numerous applications where a topical antimicrobialis useful in the treatment or prevention of infection. For example, burnwound infections remain the most common cause of morbidity and mortalityin extensively burned patients. Moreover, infection is the predominantdeterminant of wound healing, incidence of complications, and outcome ofburn patients. The main organisms responsible are Pseudomonasaeruginosa, S. aureus, Streptococcus pyogenes, and various gram-negativeorganisms. Frequent debridements and establishment of an epidermis or asurrogate, such as a graft or a skin substitute, is essential forprevention of infection. Preferably, the antimicrobial peptideformulations, alone or in combination with antibiotics, is applied toburn wounds as a gel, ointment or cream, and/or administeredsystemically. Topical application may prevent systemic infectionfollowing superficial colonization or eradicate a superficial infection.The antimicrobial peptide composition is preferably administered as a0.5 to 2% gel, cream, or ointment. Application to the skin could be doneonce a day or as often as dressings are changed. Systemic administrationcould be via intravenous, intramuscular or subcutaneous injections orinfusions. Other routes of administration known in the art could also beused.

Another use for the present compositions and methods would be in thetreatment of surgical wounds, especially those associated with foreignmaterial (e.g., sutures). Nosocomial infections may occur in as many as71% of all surgical patients, and 40% of those are infections at theoperative site. Despite efforts to prevent infection, it is estimatedthat between 500,000 and 920,000 surgical wound infections complicatethe approximately 23 million surgical procedures performed annually inthe United States. The infecting organisms are varied, but Staphylococcispp. are important organisms in these infections. Preferably, theantimicrobial peptide formulations, alone or in combination withantibiotics, is applied as an gel, ointment, cream or liquid to thewound site, or as a liquid in the wound prior to and during closure ofthe wound. Following closure, the antimicrobial peptide compositioncould also be applied at dressing changes. For surgical or trauma woundsthat are infected, the antimicrobial peptide formulation describedherein may be applied topically and/or systemically.

Yet another example, sterile gauze dressing has been the standard ofcare in catheterization for many years, but it has also beendemonstrated that transparent polyurethane film dressings are superiorbecause they permit continuous inspection of the catheterization site,they secure the device reliably, and they permit the patients to batheand shower without saturating the dressing (Maki, 1994). Therefore,certain embodiments of this invention include antimicrobial cationicpeptide compositions as described herein for use with sterile gauze orpolyurethane film dressings.

Additionally, the compositions and methods of the present invention maybe used to reduce the risk of device-related infections by directlycoating a medical device prior to insertion at a target site or byimpregnating the external surface of a medical device at the time ofmanufacture. In yet another aspect of this invention, the formulationincludes an antimicrobial cationic peptide suitable for impregnating orcoating a medical device. Thus, antimicrobial cationic peptides may beformulated as a coating or impregnation material suitable for treatingthe surfaces of a medical device or its components. In certainembodiments, such coatings and impregnation materials may includecovalent and/or non-covalent attachment of an antimicrobial cationicpeptide and analog or derivative thereof, to the interior and/orexterior surfaces of a medical device or its components. In otherembodiments, such a coating and impregnation material may include theentrapment of an antimicrobial cationic peptide in a hydrogel layer or abioerodable layer.

Other embodiments include use of antimicrobial cationic peptidecoatings, gels, ointments, and impregnation compositions alone or inconjunction with filter units or catheter components used with, forexample, a hemodialysis apparatus. In one embodiment, an arteriovenousshunt, such as a Scribner shunt, may be impregnated, coated, or adaptedto a filter containing an antimicrobial cationic peptide. Otherembodiments include the same coatings, gels, ointments, and impregnationcompositions for use with an arteriovenous fistula. In still anotherembodiment, a coating may be suitable for use with a woven fibervascular shunt.

Still other embodiments include use of antimicrobial cationic peptideformulations and methods with temporary access sites used to insert amedical device. In one preferred embodiment, antimicrobial cationicpeptide formulations may be used during a femoral vein catheterization.Other preferred embodiments include use of the antimicrobial cationicpeptide formulations with catheters, such as vascular dialysiscatheters, pulmonary artery catheters, peritoneal dialysis catheters,umbilical catheters, and subclavian vein catheters.

By way of example and not limitation, both local and systemic infectionmay result from contaminated intravascular devices, such as a CVC, andthe organisms typically responsible are coagulase-negative Staphylococci(CoNS), Staphylococcus aureus, Enterococcus spp, E. coli and Candidaspp. Hence, the antimicrobial cationic peptide or analog or derivativethereof, preferably in the form of a gel or cream, may be applied to thecatheter site prior to insertion of the catheter and then again at eachdressing change. Preferably, the peptide is at a concentration rangingfrom about 0.85% to about 1.15%. Therefore, in a typical embodiment, acomposition contains an antimicrobial cationic peptide at aconcentration ranging from about 0.01% to about 10%; aviscosity-increasing agent selected of dextran, polyvinylpyrrolidone,hydroxyethyl cellulose, or hydroxypropyl methylcellulose; and a solventof water, glycerin, propylene glycol, isopropanol, ethanol, or methanol;and at a pH ranging from about 3 to about 8.

In a preferred embodiment, the present invention is useful in a methodfor reducing microflora at a target site, comprising applying to thetarget site a composition comprising an antimicrobial cationic peptide,a viscosity-increasing agent, and a solvent. As used herein, a targetsite is any site on a subject where there is present, or there is a riskof, a primary or secondary or opportunistic infection (which infectionis outside or inside the subject), and is any site where a formulationof the present invention may be administered or applied. In certainembodiments, the microflora being reduced at the target site may beprokaryotic, eukaryotic, or viral, and preferably is prokaryotic. Inother embodiments, the method for reducing microflora at a target site,comprises applying to the target site a composition containing anantimicrobial cationic peptide, a viscosity-increasing agent, and asolvent, and further comprises inserting a medical device at the targetsite before and/or after applying the composition. In anotherembodiment, the composition may further contain a buffering agent asdescribed above and may have a pH ranging from about 3.5 to about 7. Inaddition, the composition may further contain a preservative, such asbenzoic acid, benzyl alcohol, phenoxyethanol, methylparaben,propylparaben, and the like. Preferably, the peptide is an indolicidinor analog or derivative thereof, as described herein.

Another preferred embodiment is a composition comprising (a) anantimicrobial cationic peptide wherein the cationic peptide is a peptideof up to 35 amino acids comprising one of the following: 11B7CN,11B32CN, 111B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN,11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN,11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11 J58CN, 11J67CN, 11J68CN,Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN; (b) aviscosity-increasing agent wherein the viscosity-increasing agent ishydroxyethyl cellulose at a concentration of about 1.2% to about 1.8%;(c) a buffer wherein the buffer is lactate at a concentration rangingfrom about 4 mM to about 6 mM; (d) a solvent wherein the solventcomprises glycerin at a concentration ranging from about 9% to about 11%and water at a concentration ranging from about 85% to about 90%; and(e) a pH ranging from about 3.5 to about 7. In a more preferredembodiment, the cationic peptide is at a concentration ranging fromabout 0.8% to about 1.2%. Such compositions would be useful, forexample, for topical antisepsis prior to insertion of a device (e.g.,catheter) or prosthesis (e.g., synthetic arterial graft). In anotherembodiment, the composition may be applied to a target site toameliorate inflammation, such as inflammation associated with animplanted or indwelling medical device.

The compositions and methods of the present invention would betherapeutically effective in treating or preventing acne, includingsevere acne vulgaris. Acne is due to colonization and infection of hairfollicles and sebaceous cysts by Propionibacterium acne. Most casesremain mild and do not lead to scarring, although a subset of patientsdevelop large inflammatory cysts and nodules, which may drain and resultin significant scarring. The peptide formulations as described hereinmay be incorporated into soap, or applied topically as a cream, lotion,or gel to the affected areas. The peptide formulation may be appliedeither once a day or multiple times during the day, and the length oftreatment may be for as long as the lesions are present or to preventrecurrent lesions. Alternatively, the peptide composition may beformulated to be administered orally or systemically to treat or preventacne lesions. Preferably the peptide composition is formulated fortopical administration or application. A preferred embodiment is acomposition comprising an antimicrobial cationic peptide, a bufferingagent, and a solvent; more preferably a composition, comprising (a) anantimicrobial cationic peptide wherein the cationic peptide is a peptideof up to 35 amino acids comprising one of the following: 11B7CN,11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN,11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN,11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN,Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN; (b) abuffer wherein the buffer is lactate at a concentration ranging fromabout 4 mM to about 6 mM; (c) a solvent wherein the solvent comprisesethanol at a concentration ranging from about 45% to about 55% and waterat a concentration ranging from about 44% to about 54%; and (d) a pHranging from about 3.5 to about 7. In a more preferred embodiment, thecationic peptide is at a concentration ranging from about 0.8% to about1.2%. In another preferred embodiment, the peptide composition mayfurther comprise an acne medicament such as retinoid, vitamin D3, orcorticosteroid, and analogs or derivatives thereof. Therefore, incertain preferred methods to reduce microflora, or to treat or preventan infection, at a target site, the target site may be skin, and theskin may further comprise acne.

Another example of the therapeutic value of the compositions and methodsof the present invention would be in the treatment of nosocomialinfections. For example, infection by S. aureus may result inimpetigenous lesions or infected wounds, and is associated withincreased infection rates following cardiac surgery, hemodialysis,orthopedic surgery and neutropenia, both disease induced and iatrogenic.Nasal and extra-nasal carriage of Staphylococci spp. can result inhospital outbreaks of the same Staphylococci strain that is colonizing apatient's or a hospital worker's nasal passage or extra-nasal site. Muchattention has been paid to the eradication of nasal colonization, butthe results of treatment have been generally unsatisfactory. The use oftopical antimicrobial substances, such as bacitracin, tetracycline, andchlorhexidine, results in the suppression of nasal colonization, asopposed to eradication.

Accordingly, a preferred embodiment is a composition comprising anantimicrobial cationic peptide, a viscosity-increasing agent, a solvent,and a preservative; more preferably a composition comprising (a) anantimicrobial cationic peptide wherein the cationic peptide is a peptideof up to 35 amino acids comprising one of the following: 11B7CN,11B32CN, 11B36CN, 11E3CN, 11F4CN, 11F5CN, 11F12CN, 11F17CN, 11F50CN,11F56CN, 11F63CN, 11F64CN, 11F66CN, 11F67CN, 11F68CN, 11F93CN, 11G27CN,11J02CN, 11J02ACN, 11J30CN, 11J36CN, 11J58CN, 11J67CN, 11J68CN,Nt-acryloyl-11B7CN, Nt-glucosyl-11J36CN, or Nt-glucosyl-11J38CN; (b) aviscosity-increasing agent wherein the viscosity-increasing agent ishydroxyethyl cellulose at a concentration of about 1.2% to about 1.8%;(c) a solvent wherein the solvent comprises glycerin at a concentrationranging from about 9% to about 11% and water at a concentration rangingfrom about 85% to about 90%; (d) a preservative wherein the preservativeis benzoic acid at a concentration ranging from about 20 mM to about 30mM; and (e) a pH ranging from about 3.5 to about 4.7. In a morepreferred embodiment, the cationic peptide is at a concentration rangingfrom about 0.8% to about 1.2%, or ranging from about 2.5% to about 3.5%.

These preferred compositions may be used in a method for reducingmicroflora, or for treating or preventing infection, at a target site byapplying to the target site the antimicrobial cationic peptideformulations described herein. In another embodiment, the target sitemay be a mucosa, preferably the mucosa of the nasal passage or anteriornaris.

Pharmaceutical compositions of the present invention are administered ina manner appropriate to the infection or disease to be treated. Theamount and frequency of administration will be determined by factorssuch as the condition of the patient, the cause of the infection, andthe severity of the infection. Appropriate dosages may be determined byclinical trials, but will generally range from about 0.1 to 50 mg/kg.

In addition, the compositions of the present invention may be used inthe manner of common disinfectants or in any situation in whichmicroorganisms are undesirable. For example, these peptides may be usedas surface disinfectants, coatings, including covalent bonding, formedical devices, coatings for clothing, such as to inhibit growth ofbacteria or repel mosquitoes, in filters for air purification, such ason an airplane, in water purification, constituents of shampoos andsoaps, food preservatives, cosmetic preservatives, media preservatives,herbicide or insecticides, constituents of building materials, such asin silicone sealant, and in animal product processing, such as curing ofanimal hides.

The antimicrobial cationic peptides, particularly the labeled analogsand derivatives thereof, may be used in image analysis and diagnosticassays or for targeting sites in multicellular and single cellularorganisms. As a targeting system, the analogues may be coupled withother peptides, proteins, nucleic acids, antibodies, chemical compounds(e.g., fluorescent tags), and the like.

The following Examples are provided by way of illustration and not byway of limitation.

EXAMPLES Example 1 Synthesis Purification and Characterization ofCationic Peptides and Analogues

Peptide synthesis is based on the standard solid-phase Fmoc protectionstrategy. The instrument employed is a 9050 Plus PepSynthesiser(PerSeptive BioSystems, Inc.). Polyethylene glycol polystyrene (PEG-PS)graft resins are employed as the solid phase derivatized with anFmoc-protected amino acid linker for C-terminal amide synthesis. HATU(O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) is used as the coupling reagent. During synthesis,coupling steps are continuously monitored to ensure that each amino acidis incorporated in high yield. The peptide is cleaved from thesolid-phase resin using trifluoroacetic acid and appropriate scavengersand the crude peptide is purified using preparative reversed-phasechromatography. Typically the peptide is prepared as thetrifluoroacetate salt, but other salts, such as acetate, chloride andsulfate, can also be prepared by salt exchange.

All peptides are analyzed by mass spectrometry to ensure that theproduct has the expected molecular mass. The product should have asingle peak accounting for >95% of the total peak area when subjected toanalytical reversed-phase high performance liquid chromatography(RP-HPLC), a separation method that depends on the hydrophobicity of thepeptide. In addition, the peptide should show a single band accountingfor >90% of the total band intensity when subjected to acid-urea gelelectrophoresis, a separation method based on the charge to mass rationof the peptide.

Peptide content, the amount of the product that is peptide rather thanretained water, salt or solvent, is measured by quantitative amino acidanalysis, free amine derivatization or spectrophotometric quantitation.Amino acid analysis also provides information on the ratio of aminoacids present in the peptide, which assists in confirming theauthenticity of the peptide.

Peptide analogues and their names are listed below. In this list, andelsewhere, the amino acids are denoted by the one-letter amino acid codeand lower case letters represent the D-form of the amino acid.

TABLE 1Indolicidin analogs and derivatives thereof and Other AntimicrobialCationic Peptides Apidaecin IA (SEQ ID NO: 1)G N N R P V Y I P Q P R P P H P R I Deber A2KA2  (SEQ ID NO: 2)K K A A A K A A A A A K A A W A A K A A A K K K K 10 (SEQ ID NO: 3)I L P W K W P W W P W R R 10CN (SEQ ID NO: 4) I L P W K W P W W P W R R11 (SEQ ID NO: 5) I L K K W P W W P W R R K 11CN (SEQ ID NO: 6)I L K K W P W W P W R R K 11CNR (SEQ ID NO: 7) K R R W P W W P W K K L I11A1CN (SEQ ID NO: 8) I L K K E P F F P F R R K 11A2CN (SEQ ID NO: 9)I L K K I P I I P I R R K 11A3CN (SEQ ID NO: 10)I L K K Y P Y Y P Y R R K 11A4CN (SEQ ID NO: 11) I L K K W P W P W R R K11A5CN (SEQ ID NO: 12) I L K K Y P W Y P W R R K 11A6CN (SEQ ID NO: 13)I L K K F P W F P W R R K 11A7CN (SEQ ID NO: 14)I L K K F P F W P W R R K 11A8CN (SEQ ID NO: 15) I L R Y V Y Y V Y R R K11A9CN (SEQ ID NO: 16) I L R W P W W P W W P W R R K 11A10CN(SEQ ID NO: 17) W W R W P W W P W R R K 11B1CN (SEQ ID NO: 18)I L R R W P W W P W R R K 11B2CN (SEQ ID NO: 19) I L R R W P W W P W R K11B3CN (SEQ ID NO: 20) I L K W P W W P W R R K IIB4CN (SEQ ID NO: 21)I L K K W P W W P W R K 11B5CN (SEQ ID NO: 22) I L K W P W W P W R K11B7CN (SEQ ID NO: 23) I L R W P W W P W R R K 11B7CNR (SEQ ID NO: 24)K R R W P W W P W R L I 11B8CN (SEQ ID NO: 25) I L W P W W P W R R K11B9CN (SEQ ID NO: 26) I L R R W P W W P W R R R 11B10CN (SEQ ID NO: 27)I L K K W P W W P W K K K 11B16CN (SEQ ID NO: 28)I L R W P W W P W R R K I M I L K K A G S 11B17CN (SEQ ID NO: 29)I L R W P W W P W R R K M I L K K A G S 11B18CN (SEQ ID NO: 30)I L R W P W W P W R R K D M I L K K A G S 11B19CN (SEQ ID NO: 31)I L R W P W R R W P W R R K 11B20CN (SEQ ID NO: 32)I L R W P W W P W R R K I L M R W P W W P W R R K M A A 11B32CNR(SEQ ID NO: 33) K R K W P W W P W R L I 11B36CN (SEQ ID NO: 34)I L K W V W W V W R R K 11C3CN (SEQ ID NO: 35) I L K K W A W W P W R R K11C4CN (SEQ ID NO: 36) I L K K W P W W A W R R K 11C5CN (SEQ ID NO: 37)W W K K W P W W P W R R K 11D1CN (SEQ ID NO: 38) L K K W P W W P W R R K11D3CN (SFQ ID NO: 39) P W W P W R R K 11D4CN (SEQ ID NO: 40)I L K K W P W W P W R R K M I L K K A G S 11D5CN (SEQ ID NO: 41)I L K K W P W W P W R R M I L K K A G S 11D6CN (SEQ ID NO: 42)I L K K W P W W P W R R I M I L K K A G S 11D9M8 (SEQ ID NO: 43)W W P W R R K 11D10M8 (SEQ ID NO: 44) I L K K W P W 11D11H(SEQ ID NO. 45) I L K K W P W W P W R R K M 11D12H (SEQ ID NO: 46)I L K K W P W W P W R R M 11D13H (SEQ ID NO: 47)I L K K W P W W P W R R I M 11D14CN (SEQ ID NO: 48)I L K K W W W P W R K 11D15CN (SEQ ID NO: 49) I L K K W P W W W R K11D18CN (SEQ ID NO: 50) W R I W K P K W R L P K W 11D19CN(SEQ ID NO: 51) C L R W P W W P W R R K 11E1CN (SEQ ID NO: 52)i L K K W P W W P W R R K 11E2CN (SEQ ID NO: 53)I L K K W P W W P W R R k 11E3CN (SEQ ID NO: 54)i L K K W P W W P W R R k 11F1CN (SEQ ID NO: 55)I L K K W V W W V W R R k 11F2CN (SEQ ID NO: 56)I L K K W P W W V W R R K 11F3CN (SEQ ID NO: 57)I L K K W V W W P W R R K 11F4CN (SEQ ID NO: 58) I L R W V W W V W R R K11F4CNR (SEQ ID NO: 59) K R R W V W W V W R L I 11F5CN (SEQ ID NO: 60)I L R R W V W W V W R R K 11F6CN (SEQ ID NO: 61)I L R W W V W W V W W R R K 11F12CN (SEQ ID NO: 62)R L W V W W V W R R K 11F17CN (SEQ ID NO: 63) R L W V W W V W R R11F50CN (SEQ ID NO: 64) R L G G G W V W W V W R 11F56CN (SEQ ID NO: 65)R L W W V V W W R R 11F63CN (SEQ ID NO: 66) R L V V W W V V R R 11F64CN(SEQ ID NO: 67) R L F V W W V F R R 11F66CN (SEQ ID NO: 68)R L V V W V V W R R 11F67CN (SEQ ID NO: 69) r L W V W W V W R R 11F68CN(SEQ ID NO: 70) R L W V W W V W R r 11F93CN (SEQ ID NO: 71)W V R L W W R R V W 11G2CN (SEQ ID NO: 72) I K K W P W W P W R R K11G3CN (SEQ ID NO: 73) I L K K P W W P W R R K 11G4CN (SEQ ID NO: 74)I L K K W W W P W R R K 11G5CN (SEQ ID NO: 75) I L K K W P W W W R R K11G6CN (SEQ ID NO: 76) I L K K W P W W P R R K 11G7CN (SEQ ID NO: 77)I L K K W P W W P W R R 11G13CN (SEQ ID NO: 78) I L K K W P W W P W K11G14CN (SEQ ID NO: 79) I L K K W P W W P W R 11G24CN (SEQ ID NO: 80)L W P W W P W R R K 11G25CN (SEQ ID NO: 81) L R W W W P W R R K 11G26CN(SEQ ID NO: 82) L R W P W W P W 11G27CN (SEQ ID NO: 83)W P W W P W R R K 11G28CN (SEQ ID NO: 84) R W W W P W R R K 11H1CN(SEQ ID NO: 85) A L R W P W V V P W R R K 11H2CN (SEQ ID NO: 86)I A R W P W W P W R R K 11H3CN (SEQ ID NO: 87) I L A W P W W P W R R K11H4CN (SEQ ID NO: 88) I L R A P W W P W R R K 11H5CN (SEQ ID NO: 89)I L R W A W W P W R R K 11H6CN (SEQ ID NO: 90) I L R W P A W P W R R K11H7CN (SEQ ID NO: 91) I L R W P W A P W R R K 11H8CN (SEQ ID NO: 92)I L R W P W W A W R R K 11H9CN (SEQ ID NO: 93) I L R W P W W P A R R K11H10CN (SEQ ID NO: 94) I L R W P W W P W A R K 11H11CN (SEQ ID NO: 95)I L R W P W W P W R A K 11H12CN (SEQ ID NO: 96) I L R W P W W P W R R A11J01CN (SEQ ID NO: 97) R R I W K P K W R L P K R 11J02CN(SEQ ID NO: 98) W R W W K P K W R W P K W 11J02ACN (SEQ ID NO: 99)W R W W K P K W R W P K W 11J30CN (SEQ ID NO: 100)W R W W K V A W R W V K W 11J36CN (SEQ ID NO: 101)W R W W K V W R W V K W 11J38CN (SEQ ID NO: 102)W R W W K V V W R W V K W 11J58CN (SEQ ID NO: 103)W (Orn) W W (Orn) V A W (Orn) W V (Om) W 11J67CN (SEQ ID NO: 104)W (Orn) W W (Orn) P (Orn) W (Orn) W P (Orn) W 11J68CN (SEQ ID NO: 105)W (Dab) W W (Dab) P (Dab) W (Dab) W P (Dab) W 21A1 (SEQ ID NO: 106)K K W W R R V L S G L K T A G P M Q S V L N K 21A2 (SEQ ID NO: 107)K K W W R R A L Q G L K T A G P A I Q S V L N K 21A10 (SEQ ID NO: 108)K K W W R R V L K G L S S G P A L S N V 22A1 (SEQ ID NO: 109)K K W W R R A L Q A L K N G L P A L I S 26 (SEQ ID NO: 110)K W K S F I K K L I S A A K K V V T T A K P L I S S 27 (SEQ ID NO: 111)K W K L F K K I G I G A V L K V L T T G L P A L I S 28 (SEQ ID NO: 112)K W K L E K K I G I G A V L K V L I T G L P A L K L T K 29(SEQ ID NO: 113) K W K S F I K K L T T A V K K V L T T G L P A L I S29A2 (SEQ ID NO: 114)K W K S F I K N L T K V L K K V V T T A L P A L I S 29A3(SEQ ID NO: 115) K W K S F I K K L T S A A K K V L T T G L P A L I S29F1 (SEQ ID NO: 116)K W K L F I K K L T P A V K K V E L T G L P A L I S 31 (SEQ ID NO: 117)G K P R P Y S P I P T S P R P I RY REWH 53A5 (SEQ ID NO: 118)R L A R I V V I R V A R Nt-Acryloyl-11B7C2N (SEQ ID NO: 119)Nt-glucosyl-11J36CN (SEQ ID NO: 120) Nt-glucosyl-11J38CN(SEQ ID NO: 121) Nt prefix = N-tenminal modification CN suffix =amidated C-terminus H suffix = homoserine at C-terminus M suffix = MAPbranched peptide R suffix = retro-synthesized peptide Orn = ornithineDab = diamino butyric acid Upper case letter = L-enantiomer amino acidLower case letter = D-enantiomer amino acid

Example 2 Synthesis of Modified Peptides

Antimicrobial cationic peptides, such as indolicidin analogs orderivatives thereof, are modified to alter the physical properties ofthe original peptide, either by use of modified amino acids in synthesisor by post-synthetic modification. Such modifications include:acetylation at the N-terminus, Fmoc-derivatized N-terminus,polymethylation, peracetylation, and branched derivatives. Peptidesmodified using the procedures described herein are listed in Table 4.

α-N-Terminal Acetylation.

Prior to cleaving the peptide from the resin and deprotecting it, thefully protected peptide is treated with N-acetylimidazole in DMF for 1 hat room temperature, which results in selective reaction at theα-N-terminus. The peptide is then deprotected/cleaved and purified asfor an unmodified peptide.

Fmoc-Derivatized α-N-Terminus.

If the final Fmoc deprotection step is not carried out, the α-N-terminusFmoc group remains on the peptide. The peptide is then side-chaindeprotected/cleaved and purified as for an unmodified peptide.

Polymethylation.

The purified peptide in a methanol solution is treated with excesssodium bicarbonate, followed by excess methyl iodide. The reactionmixture is stirred overnight at room temperature, extracted with organicsolvent, neutralized and purified as for an unmodified peptide. Usingthis procedure, a peptide is not fully methylated; methylation of 11CNyielded an average of 6 methyl groups. Thus, the modified peptide is amixture of methylated products.

Caprolactam Modification.

A purified peptide in DMF solution is cooled to 0° C. on ice withstirring. Added to the peptide solution is2-(1H-benzotriazole-1-yl)-1,1,3,3-teramethyluronium hexafluorophosphateand N-methylmorpholine; the reaction mixture is removed from the icebath and stirred for 1 h, until the reaction mix rises to roomtemperature. Water is added and the resulting caprolactam peptidesolution is purified by C8 RP-HPLC.

Peracetylation.

A purified peptide in DMF solution is treated with N-acetylimidazole for1 h at room temperature. The crude product is concentrated, dissolved inwater, lyophilized, re-dissolved in water and purified as for anunmodified peptide. Complete acetylation of primary amine groups isobserved.

Four/Eight Branch Derivatives.

The branched peptides are synthesized on a four- or eight-branched corebound to the resin. Synthesis and deprotection/cleavage proceed as foran unmodified peptide. These peptides are purified by dialysis against 4M guanidine hydrochloride then water, and analyzed by mass spectrometry.

TABLE 2 Modified Indolicidin analogs and derivatives thereof Peptidemodified Peptide name Modification 10 10A Acetylated α-N-terminus 11 11AAcetylated α-N-terminus 11CN 11ACN Acetylated α-N-terminus 11CN 11CNW1Fmoc-derivatized N-terminus 11CN 11CNX1 Polymethylated derivative 11CN11CNY1 Peracetylated derivative 11 11M4 Four branch derivative 11 11M8Eight branch derivative 11B1CN 11B1CNW1 Fmoc-derivatized N-terminus11B4CN 11B4ACN Acetylated N-terminus 11B7CN 11B7ACN AcetylatedN-terminus 11B7CN 11B7CNF12 Formylated Lys[12] 11B7 11B7Cap12Caprolactam Lys[12] 11B9CN 11B9ACN Acetylated N-terminus 11D9 11D9M8Eight branch derivative 11D10 11D10M8 Eight branch derivative 11G6CN11G6ACN Acetylated α-N-terminus 11G7CN 11G7ACN Acetylated α-N-terminus

Example 3 Antimicrobial Activity of Cationic Peptides

A cationic peptide may be tested for antimicrobial activity by using anin vitro assay as described below.

Agarose Dilution Assay

The agarose dilution assay measures antimicrobial activity of peptidesand peptide analogues. The activity is expressed as the minimuminhibitory concentration (MIC) in pg/ml of the peptide.

In order to mimic in vivo conditions, calcium and magnesium supplementedMueller Hinton broth is used in combination with a low EEO agarose asthe bacterial growth medium. Agarose, rather than agar, is used as thecharged groups in agar prevent peptide diffusion through the medium. Themedium is autoclaved, then cooled to 50-55° C. in a water bath beforeaseptic addition of a peptide solution. The same volume of differentconcentrations of peptide solution is added to the cooled, moltenagarose that is then poured into a petri plate to a depth of 3-4 mm andallowed to solidify.

The bacterial inoculum is adjusted to a 0.5 McFarland turbidity standard(PML Microbiological) and then diluted 1:10 before application on to theagarose plate. The final inoculum applied to the agarose isapproximately 10⁴ CFU in a 5-8 mm diameter spot. The agarose plates areincubated at 35-37° C. for 16 to 20 hours.

The MIC is recorded as the lowest concentration of peptide thatcompletely inhibits growth of the organism as determined by visualinspection. Representative MIC values for various peptide analoguesagainst bacteria and yeast are shown in Table 3.

TABLE 3 Activity of antimicrobial cationic peptides as determined byagarose dilution susceptibility testing Minimum Inhibitory Concentration(μg/ml) Organism Strain # 11B7CN 11B32CN 11B36CN 11E3CN 11F4CN A.calcoaceticus ACA002 4 2 2 4 4 E. cloacae ECL007 128 128 64 >128 64 E.coli ECO005 16 16 8 8 4 K. pneumoniae KPN001 128 32 8 32 4 P. aeruginosaPAE004 128 32 32 64 32 S. maltophilia SMA002 16 32 8 64 16 S. marcescensSMS003 >128 >128 >128 >128 >128 E. faecalis EFS001 64 64 16 64 8 S.aureus SAU014 4 8 2 8 4 S. epidermidis SEP010 4 2 2 4 2 S. mitis SMT0142 2 2 2 2 S. pneumoniae SPN002 4 8 4 4 8 S. pyogenes SPY001 1 2 2 1 2 C.jeikeium CJK005 0.5 0.5 1 0.5 2 C. albicans CAL002 16 16 8 32 32 C.neoformans CNE001 4 4 16 4 8 Minimum Inhibitory Concentration (μg/ml)Organism Strain # 11F5CN 11F12CN 11F17CN 11F50CN 11F27CN 11F56CN A.calcoaceticus ACA002 4 1 1 2 2 1 E. cloacae ECL007 128 32 16 >128 128 32E. coli ECO005 16 4 2 4 16 2 K. pneumoniae KPN001 64 8 2 16 32 2 P.aeruginosa PAE004 64 16 16 >128 32 32 S. maltophilia SMA002 16 2 1 8 8 2S. marcescens SMS003 >128 >128 >128 >128 >128 >128 E. faecalis EFS001 162 2 16 16 2 S. aureus SAU014 8 2 1 2 8 1 S. epidermidis SEP010 2 1 1 1 21 S. mitis SMT014 2 1 1 1 4 1 S. pneumoniae SPN002 4 4 1 2 16 2 S.pyogenes SPY001 2 0.5 0.5 1 1 1 C. jeikeium CJK005 0.5 0.5 0.5 0.5 0.50.5 C. albicans CAL002 32 16 32 32 16 16 C. neoformans CNE001 8 4 4 16 48 Minimum Inhibitory Concentration (μg/ml) Organism Strain # 11F63CN11F64CN 11F66CN 11F67CN 11F68CN A. calcoaceticus ACA002 2 2 2 1 E.cloacae ECL007 16 16 8 32 32 E. coli ECO005 2 2 2 2 2 K. pneumoniaeKPN001 4 2 2 2 2 P. aeruginosa PAE004 8 16 8 16 32 S. maltophilia SMA0020.5 1 2 2 2 S. marcescens SMS003 >128 >128 >128 >128 >128 E. faecalisEFS001 4 2 2 4 2 S. aureus SAU014 2 1 2 2 2 S. epidermidis SEP010 1 1 11 1 S. mitis SMT014 2 1 1 1 1 S. pneumoniae SPN002 4 2 2 2 2 S. pyogenesSPY001 1 1 1 1 1 C. jeikeium CJK005 1 0.5 1 0.5 0.5 C. albicans CAL00216 16 16 32 32 C. neoformans CNE001 8 8 8 8 8 Minimum InhibitoryConcentration (μg/ml) Organism Strain # 11F93CN 11G27CN 11J02CN 11J02ACN11J30CN 11J36CN A. calcoaceticus ACA002 4 16 2 2 2 2 E. cloacae ECL00764 >128 128 >32 16 32 E. coli ECO005 4 64 16 16 8 8 K. pneumoniae KPN00164 >128 32 32 2 4 P. aeruginosa PAE004 64 >128 32 64 32 64 S.maltophilia SMA002 8 64 4 8 4 2 S. marcescensSMS003 >128 >128 >128 >128 >128 >128 E. faecalis EFS001 16 >128 64 64 3216 S. aureus SAU014 2 16 2 8 2 1 S. epidermidis SEP010 2 8 2 2 2 1 S.mitis SMT014 2 8 2 2 1 1 S. pneumoniae SPN002 4 32 8 16 2 2 S. pyogenesSPY001 1 2 1 1 2 2 C. jeikeium CJK005 0.5 0.5 1 0.5 0.5 0.5 C. albicansCAL002 16 32 16 32 16 16 C. neoformans CNE001 8 16 4 16 4 4 MinimumInhibitory Concentration (μg/ml) Nt-Glucosyl Nt-Glucosyl Organism Strain# 11J58CN 11J67CN 11J68CN 11J36CN 11J38CN A. calcoaceticus ACA002 1 10.5 1 2 E. cloacae ECL007 16 32 16 64 32 E. coli ECO005 2 8 8 4 16 K.pneumoniae KPN001 2 8 4 2 16 P. aeruginosa PAE004 16 8 4 16 128 S.maltophilia SMA002 2 1 4 4 16 S. marcescens SMS003 >128 >128128 >128 >128 E. faecalis EFS001 32 64 16 16 16 S. aureus SAU014 1 2 1 22 S. epidermidis SEP010 1 1 1 2 2 S. mitis SMT014 1 0.5 0.5 1 2 S.pneumoniae SPN002 2 2 1 2 2 S. pyogenes SPY001 1 <0.25 0.5 1 2 C.jeikeium CJK005 0.5 <0.25 <0.25 1 1 C. albicans CAL002 8 8 8 16 16 C.neoformans CNE001 2 2 1 4 4

Example 4 An In Vitro Drug Release Method for Topical Formulations UsingLow-Flow Cells

An in vitro drug release method for topical formulations using alow-flow cells is used to examine the release of peptide fromexperimental formulations. The cell consists of an upper (donor) chamberphysically separated from a lower (receptor) chamber by a permeablesynthetic membrane (Tuffryn, Gelman). A total of 1 gram of a candidateformulation is placed in the donor chamber. The receptor fluid(distilled water, 37° C.) is pumped through the receptor chamber at 2ml/h. Fractions were collected at various hourly intervals. Fractionswere collected into vials, and the amount of receptor fluid collected ingrams per fraction was recorded. The concentration of drug in thereceptor fluid was determined by RP-HPLC on a Nova Pak C8 column. Thecolumn was eluted with a gradient from 20% to 40% acetonitrile over 10min. using 0.1% aqueous trifluoroacetic acid, and 0.1% trifluoroaceticacid in acetonitrile, as solvents. The flow rate was 1 ml/min.

TABLE 4 Flow Cell Measurement of In Vitro Release Time Gel Gel GelPeriod (h) 73A 75A 76A 0-1 974 789 992 2-3 760 707 777 4-6 722 503 6717-9 590 414 435 10-12 541 499 455 13-15 396 400 376 16-18 303 300 24919-21 312 204 220 22-24 309 276 372The membrane is a 0.45 μm Tuffryn (hydrophilic polysulfone) membrane,the data represents μg of antimicrobial cationic peptide released duringthe time interval. The detection limit is 1 μg released per hour. Thedata in Table 4 shows that gel formulations show excellent initialrelease that should facilitate rapid antibiotic action upon application.The gel formulations also demonstrate good sustained release of drug.During the 22-24 hour collection, the average concentration of drugreleased into the receptor fluid ranged from 93 μgrams/ml (Gel 75A) to124 μgrams/ml (Gel 76A)—well above the MIC values for sensitiveorganisms.

TABLE 5 Compositions of Gels and Creams Tested Ingredient Gel 73A Gel75A Gel 76A Antimicrobial Cationic Peptide 1.0 1.0 1.0 HydroxyethylCellulose, NF 1.5 1.5 1.5 Glycerin, USP 10.0 10.0 10.0Polyvinylpyrrolidone 90 — 1.0 — 0.1M Lactate buffer, pH 4 5.0* 5.0 5.0Dextran (40,000), USP — — 1.0 Purified Water, USP 100.0 100.0 100.0 Allentries are grams added/100 grams of gel.

Example 5 Antimicrobial Activity of an Aqueous 1.0% Cationic Peptide Gel

The objective of this study was to assess the antimicrobial activity of1.0% antimicrobial cationic peptide gel against Pseudomonas aeruginosaPA004; Candida albicans CA002; Staphylococcus aureus SA016; andStaphylococcus epidermidis SE010. Briefly, a 2 g portion of 11B7CN 1.0%Gel was aseptically transferred into each of sixteen 50 mL tubes. Fourtubes, one for each bacterium, were labeled as day 0, day 1, day 3, andday 7. As a control, a 2 g portion of vehicle (gel without antimicrobialcationic peptide 1.0%) was aseptically transferred into each of sixteen50 mL tubes and labeled as described above.

An inoculum of 1×10⁸ CFU/ml for each of the above listed organisms wasprepared. For each of the series of tubes designated for each specificorganism, 10 μl of undiluted inoculum was added to the gel in each ofthe tubes giving a final bacterial concentration of 5×10⁵ CFU/g of gel.The contents of the tube were mixed using the handle of a sterile swab.

The tubes labeled days 1, 3, and 7 were held at ambient temperatures andsampled at the indicated time point. The day 0 tubes were sampledimmediately. At the time of sampling, 1 g of gel from the test orcontrol tube was removed and streaked on appropriate culture media.Growth of the appropriate organism was observed after 24 and 48 hours ofincubation. The remaining 1 g of test or control gel was added to salineand serially diluted. Aliquots of 0.1 ml were plated in duplicate onappropriate medium and counted after 24 and 48 hours of incubation.

The results of this study are described in Table 6. The 1.0%antimicrobial cationic peptide gel has activity against P. aeruginosaPA004; C. albicans CA002; S. aureus SA016; and S. epidermidis SE010.Each of the organisms was killed immediately upon exposure to the 1.0%antimicrobial cationic peptide gel.

TABLE 6 Summary of Colony Counts for Four Organisms with 1.0%Antimicrobial Cationic Peptide Gel Organism Tested: C. albicans (CA002)MIC = 64 ug/ml Formulation without 11B7CN (48 hour counts) 11B7CN inFormulation (48 hour counts) Time 0 1 Day 3 Days 7 Days Time 0 1 Day 3Days 7 Days Avg. Colony Avg. Colony Avg. Colony Avg. Colony Avg. ColonyAvg. Colony Avg. Colony Avg. Colony Count Count Count Count Count CountCount Count Dilution (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g)(CFU/g) (CFU/g) Neat TNTC TNTC TNTC TNTC 0 0 0 0 10⁻¹ 1.5 × 10⁵ TNTCTNTC TNTC 0 0 0 0 10⁻² 1.2 × 10⁵ 3.3 × 10⁵ 5.0 × 10⁵ 4.1 × 10⁵ 0 0 0 010⁻³ 2.5 × 10⁵ 3.5 × 10⁵ 5.0 × 10⁵ 7.5 × 10⁵ 0 0 0 0 10⁻⁴ 0 0 3.0 × 10⁶1.5 × 10⁶ 0 0 0 0 Organism Tested: P. aeruginosa (PA004) MIC = 128 ug/mlFormulation without Antimicrobial Antimicrobial cationic cationicpeptide (48 h counts) peptide in Formulation (48 h counts) Time 0 1 Day3 Days 7 Days Time 0 1 Day 3 Days 7 Days Avg. Colony Avg. Colony Avg.Colony Avg. Colony Avg. Colony Avg. Colony Avg. Colony Avg. Colony CountCount Count Count Count Count Count Count Dilution (CFU/g) (CFU/g)(CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) Neat 5.3 × 10³ 1.0 × 10²0 0 0 0 0 0 10⁻¹ 5.5 × 10³ 0 0 0 0 0 0 0 10⁻² 0 0 0 0 0 0 0 0 10⁻³ 0 0 00 0 0 0 0 Organism Tested: S. aureus (SA016) MIC = 2 ug/ml Formulationwithout Antimicrobial Antimicrobial cationic cationic peptide (48 hcounts) peptide in Formulation (48 h counts) Time 0 1 Day 3 Days 7Days^(a) Time 0 1 Day 3 Days 7 Days Avg. Colony Avg. Colony Avg. ColonyAvg. Colony Avg. Colony Avg. Colony Avg. Colony Avg. Colony Count CountCount Count Count Count Count Count Dilution (CFU/g) (CFU/g) (CFU/g)(CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) Neat TNTC TNTC TNTC TNTC 0 0 0 010⁻¹ TNTC TNTC TNTC TNTC 0 0 0 0 10⁻² 4.8 × 10⁵ 8.4 × 10⁵ 7.6 × 10⁵ 4.2× 10⁵ 0 0 0 0 10⁻³ 3.5 × 10⁵ 1.0 × 10⁶ 1.3 × 10⁶ 6.0 × 10⁵ 0 0 0 0Organism Tested: S. epidermidis (SE010) MIC = 4 ug/ml Formulationwithout Antimicrobial Antimicrobial cationic cationic peptide (48 hcounts) peptide in Formulation (48 h counts) Time 0 1 Day 3 Days 7Days^(a) Time 0 1 Day 3 Days 7 Days Avg. Colony Avg. Colony Avg. ColonyAvg. Colony Avg. Colony Avg. Colony Avg. Colony Avg. Colony Count CountCount Count Count Count Count Count Dilution (CFU/g) (CFU/g) (CFU/g)(CFU/g) (CFU/g) (CFU/g) (CFU/g) (CFU/g) Neat TNTC TNTC 2.1 × 10⁴ 1.2 ×10³ 0 0 0 0 10⁻¹ 8.2 × 10⁴ 5.5 × 10⁴ 1.9 × 10⁴ 3.0 × 10³ 0 0 0 0 10⁻²1.3 × 10⁵ 4.5 × 10⁴ 1.0 × 10⁴ 0 0 0 0 0 10⁻³ 1.0 × 10⁵ 5.0 × 10⁴ 0 0 0 00 0

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

1. A method for preventing an infection caused by a prokaryotic oreukaryotic organism at a target site, comprising applying to the targetsite a composition comprising an antimicrobial cationic peptide at aconcentration ranging from about 0.01% to about 10% (w/w), aviscosity-increasing agent, and a solvent, wherein the cationic peptideis a peptide of up to 35 amino acids comprising 11B7CN (SEQ ID NO: 23),the viscosity-increasing agent is hydroxyethyl cellulose at aconcentration of about 0.5% to about 5% (w/w), and the solvent is waterand glycerin, wherein the solvent comprises glycerin at a concentrationup to about 20% (w/w), and wherein the composition is a gel.